# Patent

## Title
Genetic test and genetic basis for SCID in pigs

## Abstract

The genetic basis for severe combined immunodeficiency disease (SCID) in pigs is described. In addition, tests for detecting pigs that are carriers for SCID or pigs with SCID are also described. Further, methods for producing pigs or herds of pigs with SCID are also described. Further, methods and compositions for treating, ameilioraing, inhibiting or correcting SCID are provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §119 to provisional application Ser. No. 61/925,072 filed Jan. 8, 2014, herein incorporated by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under Grant No. R24 OD19813 from the National Institutes of Health. The government has certain rights in the invention.


The instant application contains a Sequence Listing which has been submitted electronically via EFS-Web in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, Created on Mar. 27, 2015, is named Dekkers_14592586_Sequence Listing_ST25.txt and is 89,000 bytes in size.
FIELD OF THE INVENTION
The present invention relates to porcine subjects with severe combined immunodeficiency disease (SCID). The invention also relates to modification of the DNA cross-link repair 1C (DCLRE1C) gene, also referred to as the Artemis gene. The invention also relates to methods for generating pigs with SCID. The invention also relates to methods for increasing or decreasing SCID in a pig population. This invention also relates to the use of SCID pigs for investigation of diseases and conditions. This invention also relates to treating or correcting SCID in pigs by providing functional Artemis protein or a gene encoding functional Artemis protein.
BACKGROUND OF THE INVENTION
Pigs, and in particular members of the species Sus scrofa, are invaluable as a model organism. Because both the size and anatomy of domesticated pigs closely resembles that of humans, they are used in the research of a variety of important human diseases. However, the existing transgenic models of severe combined immunodeficiency disease (SCID) cannot mimic any of the known human immunodeficiency diseases where conditioning for bone marrow transplants includes irradiation. Further, none of the transgenic models represent a natural porcine model of SCID, and none of the transgenic models target the Artemis gene or any gene which causes radiosensitive immunodeficiency. Thus these transgenic models cannot mimic any of the known human immunodeficiency diseases where conditioning for bone marrow transplants includes irradiation.
SCID is a group of primary immunodeficiency disorders characterized by increased susceptibility to severe infections. SCID is caused by heritable defects of the cellular and humoral immune system, which result in a number of different phenotypes. Individuals with SCID have low numbers of circulating lymphocytes, T cell lymphopenia, and variable defects in B and NK cell populations. As a result, individuals with SCID have increased susceptibility to infections and increased mortality, commonly characterized by a severe deficiency in naïve T-cells. Infants born with SCID typically appear normal at birth, but are at high risk of serious infections after waning of maternal antibodies. If untreated, SCID has 100% mortality. Treatment is generally hematopoietic stem cell transplantation (HSCT), although gene therapy has been successfully used in some forms of SCID.
Characterization of the molecular mechanisms of SCID is integral to the development of diagnostic assays and treatments for the disease. A number of animal models for SCID exist, including several mouse models. However, murine and other small animal models often translate poorly to human disease due to important differences between mice and humans, including differences in immune response molecules and networks. Domestic pigs are much closer to humans, both physiologically and immunologically, and therefore a SCID pig represents a much better model for both the immune-compromised patient and for cancer and stem cell research, but such models have been lacking. While several reports have used molecular technologies to mutate one or more of several genes to cause a SCID phenotype in pigs, these reports are mainly limited to describing the phenotype of the mutated pigs. Further, these mutations are limited to genes that do not cause immunodeficiency syndromes that include a sensitivity to irradiation, as demonstrated for at least six types of human SCID genetic disease Previous work has also demonstrated that human cells transferred into SCID pigs are not destroyed due to lack of an immune system, but a practical model for use in exploiting SCID pigs for vaccine and other biomedical research has not been reported.
The Artemis gene—also referred to as the DNA cross-link repair 1C (DCLRE1C) gene—encodes a nuclear protein that is involved in V(D)J recombination and DNA repair. In pigs, based on build 10.2 of the swine genome, the Artemis gene is located on the forward strand of chromosome 10 and begins at approximately base number 51553277 and ends at approximately base number 51596761. Artemis includes 15 exons spanning 44 kb. The mRNA of porcine Artemis is 2388 nucleotides, encoding a protein that is 762 amino acids long. The protein has endonuclease activity on 5′ and 3′ overhangs and hairpins when complexed with and phosphorylated by DNA-dependent protein kinase catalytic subunit (PRKDC). Artemis is a member of the SNM1 family, which is defined by homology to yeast SNM1. Artemis is also referred to as SNM1C. Artemis is responsible for the resolution of hairpin coding ends in V(D)J recombination. In DNA double-strand break repair, Artemis is implicated in the end-processing step of the non-homologous end-joining (NHEJ) pathway. Artemis is the nuclease required for the resolution of hairpin coding ends during V(D)J recombination, the process by which B cell antibody genes and T cell receptor genes are assembled from individual V (variable), D (diversity), and J (joining) segments. For example, in joining a V segment to a D segment, the RAG (recombination activating gene) nuclease cuts both DNA strands adjacent to a V segment and adjacent to a D segment. The intervening DNA between the V and D segments is ligated to form a circular DNA molecule that is lost from the chromosome. At each of the two remaining ends, called the coding ends, the two strands of DNA are joined to form a hairpin structure. Artemis nuclease, in a complex with PRKDC, binds to these DNA ends and makes a single cut near the tip of the hairpin. The exposed 3′ termini are subject to deletion and addition of nucleotides by a variety of exonucleases and DNA polymerases, before the V and D segments are ligated to restore the integrity of the chromosome. The exact site of cleavage of the hairpin by Artemis is variable, and this variability, combined with random nucleotide deletion and addition, confers extreme diversity upon the resulting antibody and T-cell receptor genes, thus allowing the immune system to mount an immune response to virtually any foreign antigen.
In Artemis-deficient individuals, V(D)J recombination is blocked because the hairpin ends cannot be opened, and so no mature B or T cells are produced, resulting in SCID. Artemis was first identified as the gene defective in a subset of SCID patients that were unusually sensitive to radiation. Cells deficient in Artemis are more sensitive than normal cells to X-rays and to chemical agents that induce double-strand breaks (DSBs), and they show a higher incidence of chromosome breaks following irradiation. Artemis-deficient patients are also among those patients with poorer than average outcomes after bone marrow transplantation, thus a good large animal model that would recapitulate these defects would be useful for testing better methods of bone marrow transplant treatments.
The inventors have identified pigs with SCID, and a novel genetic basis of SCID in pigs. The inventors have further identified the genomic region that harbors the causative mutation, and have developed genetic marker tests that can be used to identify SCID pigs and SCID carriers. Specifically, the inventors provide a pig that possesses a mutated Artemis gene, resulting in the decreased production and/or function of the Artemis protein gene product.
It is an object of the present invention to provide the molecular basis for non-induced SCID in pigs.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects with SCID.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a model of human SCID for biomedical research.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a xenograft recipient in cancer research.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a xenograft recipient in stem cell research.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a xenograft recipient in vaccine research.
It is a further object of the present invention to provide a genetic test for determining whether a porcine subject has SCID.
It is a further object of the present invention to provide a genetic test that can be used to identify affected pigs or carriers for the defect in pig populations, including commercial populations.
It is a further object of the present invention to provide a genetic test can be used to identify SCID affected piglets at an early age.
It is a further object of the present invention to provide a genetic test that can be used in biomedical research.
It is a further object of the present invention to provide further characterization of the SCID phenotype for animal disease and biomedical research, including research into the immune system, cancer research, the effects of disease, cell and tissue transplantation, and for testing of new vaccines and therapeutic agents for immuno-compromised individuals.
It is yet another object of the invention to provide further information for understanding SCID in pigs.
It is yet another object of the present invention to provide methods of identifying other mutations that are in linkage disequilibrium with or that are causative of SCID in specific lines, populations, or breeds of pigs.
It is a further object of the present invention to provide methods and compositions for curing, treating, alleviating, or inhibiting SCID.
It is a further object of the present invention to provide pharmaceutical compositions for curing, treating, alleviating, or inhibiting SCID.
Other objects will become apparent from the detailed description of the invention which follows.
BRIEF SUMMARY OF THE INVENTION
In its broadest aspect, the present invention provides methods for identifying porcine subjects with SCID. This can be used to select for and create porcine subjects and herds of porcine subjects with SCID as an animal model to study the disease. In a further aspect, the identified and selected porcine subjects and herds of porcine subjects of the invention have SCID resulting from mutation of the genomic sequence of the Artemis gene. In an even further aspect of the invention, the porcine subjects and herds of porcine subjects of the invention have SCID resulting from mutations that modulate the expression or function of the Artemis protein gene product. According to the invention, the inventors have discovered the basis for SCID in porcine, namely mutations that inactivate the Artemis gene. According to the invention, two independent mutations in the Artemis gene result in inactivation of the Artemis gene, when present in homozygous form or as a compound heterozygote. The Inventors have also discovered that pigs harboring these mutations in homozygous or compound heterozygous form show extremely decreased numbers of T and B lymphocytes, as well as circulating antibody, and significantly abnormal thymus and lymph nodes.
In one aspect, the invention includes a genetic test for identifying SCID pigs and SCID carriers. The genetic test can be used to identify carriers for the defect in commercial pig populations, for example to eliminate the identified mutations from breeding populations and thereby eliminate the possibility of creating litters with SCID piglets which after weaning will always die from disease commonly found on commercial farms. The genetic test can also be used to identify SCID affected piglets at an early age. The genetic test can also be used to identify and create an animal model and population of the same for use in biomedical research. Identification and understanding of the genetic basis for the SCID mutation will allow further characterization of the SCID phenotype for animal disease and biomedical research, including research into the immune system, cancer research, the effects of disease, cell and tissue transplantation, and for testing of new vaccines and therapeutic agents for immuno-compromised individuals. SCID pigs provide a much better animal model for such purposes than SCID mice and, thus, development of this model could potentially be extremely valuable.
The invention also provides novel coding sequences that relate to SCID. The mutations of the Artemis gene have been found to decrease expression and/or function of the Artemis protein gene product. The mutant gene and protein allow for the development of in vitro and in vivo models and agents to improve therapies for SCID.
In another aspect, the invention provides methods for generating SCID in animals, including pigs. The methods can be used to target the Artemis gene in a pig or herd of pigs to generate a population of pigs that are affected by SCID or are carriers of SCID. In another aspect, the methods can be used to improve the production of SCID pigs, for example by conducting bone marrow transplants on homozygous mutant SCID pigs, thereby allowing for mating of homozygous mutant SCID pigs to heterozygous carrier pigs, resulting in 50% affected piglets compared to 25% from carrier by carrier matings.
In another aspect of the invention, one may use the Artemis gene to screen for other markers in linkage disequilibrium with the mutation of the invention to create further tests, to identify other potential SCID associated mutations in other species of meat animals, lines, populations, or breeds of the same.
In another aspect of the invention, one may use the polynucleotide and polypeptide sequences provided herein to inhibit, treat, or ameliorate SCID by introducing a heterologous Artemis protein or nucleic acid encoding the same. In one aspect, the invention encompasses pharmaceutical compositions comprising a heterologous Artemis protein or nucleic acid encoding the same.
In another aspect of the invention, one may utilize gene repair mechanisms, including gene editing, to repair defects in the Artemis gene. In one embodiment, these methods may be used to cure, treat, or ameliorate SCID in an individual or animal.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows absence of B and T cells in peripheral blood of SCID pigs, measured by flow cytometry. Representative plots were first gated on lymphocyte characteristics, and then the lymphocyte population was characterized by specific antibodies detecting cell-type specific surface proteins. Antibodies specific to CD3 (T cells), CD21 (B cells), and CD16 (NK cells), were used to detect these proteins and enumerate the proportion of lymphocytes consisting of T, B and NK cells.
FIG. 2 shows quantitative PCR analysis of NCR1 (NK cells), CD3E (T cells), and CD79A (B cells) expression, demonstrating very low levels of B and T marker gene expression in suspect SCID pig whole blood (p<0.01) compared to normal littermates.
FIG. 3 shows immunoglobulin (Ig) measured in umbilical cord blood from SCID piglets versus normal littermates, demonstrating the lack of antibodies in newborn SCID pigs (#2, 7, 10, 14, 15) compared to non-SCID pigs (all other data).
FIG. 4 shows segregation of the SCID phenotype as a recessive autosomal mutation. The 4 dams and 2 sires produced the initial 4 SCID piglets.
FIG. 5 shows Genome-Wide Association results for the SCID phenotype, showing a region on porcine chromosome 10 to be associated with the SCID phenotype. The solid black line shows the position of the Artemis gene.
FIG. 6 shows the 12 and 16 haplotypes that are associated with the SCID phenotype. Sire 15801 carries the 12 haplotype and sire 11403 and the 4 dams that produced the initial four SCID piglets (see FIG. 4) carry the 16 haplotype. The outlined SNPs were used to design allele specific primers, as described in section 4a.
FIG. 7(A-E) shows the SCID pedigree, tracing the 12 and 16 haplotypes that were identified in generation 8 back to the founder generation of the population.
FIG. 8(A-C) shows presence or absence of exons in transcripts sequenced from haplotype 16 (A), haplotype 12 (C), and normal (B) using Full cDNA primers (SEQ ID NO:39 and 40). Exon 8 is absent in all haplotype 16 sequences, while exon 10 is absent in all haplotype 12 sequences. Light pink boxes indicate splicing of partial exons, with numbers showing the number of bases present for that exon. Purple boxes indicate that the transcript included 167 bases of intron 4 after exon 4.
FIG. 9 shows SCID cells are highly sensitive to radiation compared to cells from normal pigs. Fibroblasts from affected and unaffected animals (n=7-10) were subjected to gamma rays at 0, 2, 4, or 8 Grays. Letters denote significant differences between genotypes within gamma radiation level (p<0.0001).
FIG. 10 shows design for the use of Bone Marrow Transfer to produce homozygous SCID pigs for breeding.
FIG. 11 shows evidence of recovery of blood lymphocyte counts in Bone Marrow Transfer SCID recipients.
FIG. 12(A-B) shows evidence of donor increasing donor origin of PBMC's in female Bone Marrow Transfer recipients that received bone marrow cells from male donors. Assays based on presence of SRY sequences.
FIG. 13 shows evidence of an active adaptive immune system in Bone Marrow Transfer SCID recipients. Serum antibody to vaccination with Circumvent: Circovirus-Mycoplasma.
FIG. 14(a-c) shows two independent mutations in Artemis causing SCID. (a) The exons are shown as blocks, the coding region of the Artemis transcript is indicated by cross-hatch within these blocks, and the normal, h12, and h16 genotypes at mutated positions are shown. Mutant alleles that cause SCID are shown by pink dots. (b) Genomic sequence of h16 shows a splice donor site mutation (g→a) responsible for the lack of exon 8 in all h16 transcripts. (c) A point mutation (G→A) in exon 10 changes the Tryptophan at position 267 to a stop codon in h12.
FIG. 15 shows rescue of sensitivity to ionizing radiation. Fibroblasts from SCID (n=2) and normal (n=3) pigs were transfected with 5 μg Artemis, 3.45 μg Exodus plasmid without Artemis gene, or were shocked without plasmid added. Fibroblasts were exposed to 4 Gy irradiation dose 24 hours after transfection. Colonies were counted after 14 days of growth. Error bars represent the standard error of the least squares means. Dots show individual observations. Different letters within affected status represent statistical differences between means with p<0.01.
FIG. 16(A-X) shows sequences used in the present invention. In each nucleic acid sequence, primer sequences are shown in bold, correct translation frames are underlined, location of missing sequences are indicated in parentheses, and premature stop codon are shown in italics. Splice donor site mutation indicated by brackets. Point mutations indicated by curly brackets.



Various embodiments of the present invention will be described in detail with reference to the drawings, wherein like reference numerals represent like parts throughout the several views. Reference to various embodiments does not limit the scope of the invention. Figures represented herein are not limitations to the various embodiments according to the invention and are presented for exemplary illustration of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention now will be described more fully with reference to the accompanying examples. The invention may be embodied in many different forms and these embodiments should not be construed as limited to the embodiments set forth in this application; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.
Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains, having the benefit of the teachings presented in the descriptions and the drawings herein. As a result, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used in the specification, they are used in a generic and descriptive sense only and not for purposes of limitation.
Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e. g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
As used herein, “antisense orientation” includes reference to a duplex polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid.
One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine; and UGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made.
Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for its native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins W. H. Freeman and Company.
By “encoding” or “encoded”, with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise intervening sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed.
As used herein “full-length sequence” in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of a native (nonsynthetic), endogenous, biologically active form of the specified protein. Methods to determine whether a sequence is full-length are well known in the art including such exemplary techniques as northern or western blots, primer extension, S1 protection, and ribonuclease protection. Comparison to known full-length homologous (orthologous and/or paralogous) sequences can also be used to identify full-length sequences of the present invention. Additionally, consensus sequences typically present at the 5′ and 3′ untranslated regions of mRNA aid in the identification of a polynucleotide as full-length. For example, the consensus sequence ANNNNAUGG, where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5′ end. Consensus sequences at the 3′ end, such as polyadenylation sequences, aid in determining whether the polynucleotide has a complete 3′ end.
As used herein, “heterologous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
By “host cell” is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.
The term “hybridization complex” includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
The term “introduced” in the context of inserting a nucleic acid into a cell is equivalent to “transfection” or “transformation” or “transduction,” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e. g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment—the isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native that material. The alteration to yield the synthetic material can be performed on the material within, or removed from its natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced by nonnaturally occurring means to a locus of the genome not native to that nucleic acid. Nucleic acids which are “isolated” as defined herein, are also referred to as “heterologous” nucleic acids.
As used herein, “localized within the chromosomal region defined by and including” with respect to particular markers includes reference to a contiguous length of a chromosome delimited by and including the stated markers. Where applicable, the location of the sequences within the porcine genome assembly (http://useast.ensembl.org/Sus_scrofa/Location/Genome) is provided.
As used herein, “marker” includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A “polymorphic marker” includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes of that pair to be followed. A genotype may be defined by use of one or a plurality of markers.
As used herein, “mutation” includes reference to alterations in the nucleotide sequence of a polynucleotide, such as for example a gene or coding DNA sequence (CDS), compared to the wild-type sequence. The term includes, without limitation, substitutions, insertions, frameshifts, deletions, inversions, translocations, duplications, splice-donor site mutations, point-mutations or the like.
As used herein, “nucleic acid” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses conservatively modified variants and known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids).
By “nucleic acid library” is meant a collection of isolated DNA or RNA molecules which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).
As used herein “operably linked” includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or conservatively modified variants; the term may also refer to analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also may apply to conservatively modified variants and to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “polypeptide”, “peptide” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitization, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Further, this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention.
As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.
As used herein “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
As used herein, a “recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
The term “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to another nucleic acid sequence or other biologics. When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a reference nucleic acid sequence, and then by selection of appropriate conditions the probe and the reference sequence selectively hybridize, or bind, to each other to form a duplex molecule.
The term “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing).
Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e. g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA/DNA hybrids, the thermal melting point (Tm) can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138: 267-284 (1984): Tm [° C.]=81.5+16.6 (log M)+0.41(% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1 to 4° C. lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6 to 10° C. lower than the Tm; low stringency conditions can utilize a hybridization and/or wash at 11 to 20° C. lower than the Tm. Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
As used herein, “transgenic animal, cell or tissue” includes reference to an animal which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to include any cell, cell line, tissue, or organ, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional breeding methods or by naturally occurring events such as random cross-fertilization, non recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. “Mutation” as used herein includes without limitation, deletions, inversions, translocations, duplications, splice-donor site mutations, point-mutations or the like.
As used herein, “vector” includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
The following terms are used to describe the sequence relationships between a polynucleotide/polypeptide of the present invention with a reference polynucleotide/polypeptide: (a)“reference sequence”, (b)“comparison window”, (c) “sequence identity”, and (d) “percentage of sequence identity”.
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison with a polynucleotide/polypeptide of the present invention. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” includes reference to a contiguous and specified segment of a polynucleotide/polypeptide sequence, wherein the polynucleotide/polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide/polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides/amino acids residues in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide/polypeptide sequence, a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); and by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA, and related programs in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). The CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994).
The BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Altschul et al., J. Mol. Biol., 215: 403-410 (1990); and, Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Software for performing BLAST analyses is publicly available, for example through the National Center for Biotechnology Information (ncbi.nlm.nih.gov/). This algorithm has been thoroughly described in a number of publications. See, e.g., Altschul S F et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, 25 NUCLEIC ACIDS RES. 3389 (1997); National Center for Biotechnology Information, THE NCBI HANDBOOK [INTERNET], Chapter 16: The BLAST Sequence Analysis Tool (McEntyre J, Ostell J, eds., 2002), available at http://www.ncbi.nlm.nih.gov/books/NBK21097/pdf/ch16.pdf. The BLASTP program for amino acid sequences has also been thoroughly described (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5877 (1993)). A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17: 191-201 (1993)) low-complexity filters can be employed alone or in combination.
Unless otherwise stated, nucleotide and protein identity/similarity values provided herein are calculated using GAP (GCG Version 10) under default values. GAP (Global Alignment Program) can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP represents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).
Multiple alignment of the sequences can be performed using the CLUSTAL method of alignment (Higgins and Sharp (1989) CABIOS. 5: 151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the CLUSTAL method include KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
(c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions may be calculated according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988), for example as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, the term “porcine” refers to species which are part of the Sus genus of the Suidae family. This includes domestic pigs of any breed. Thus, any of the various pig, swine, or hog species, whether male or female, are included in the term, and adult, fetal and new-born animals are intended to be covered. The term does not denote a particular age. One example of a porcine subject is a member of the Yorkshire breed pig population.
The term “genetic marker” refers to a variable nucleotide sequence (polymorphic) that is present in porcine genomic DNA on a chromosome and that is identifiable with specific oligonucleotides. For example, such a variable nucleotide sequence is distinguishable by nucleic acid amplification and observation of a difference in size or sequence of nucleotides due to the polymorphism. In useful embodiments, such genetic markers may be identified by several techniques known to those skilled in the art, and include typing of microsatellites or short tandem repeats (STR), restriction fragment length polymorphisms (RFLP), detection of deletion or insertion sites, and random amplified polymorphic DNA (RAPD) as well as the typing of single nucleotide polymorphism (SNP) by methods including restriction-fragment-length polymerase chain reaction, allele-specific oligomer hybridization, oligomer-specific ligation assays, mini-sequencing, direct sequencing, fluorescence-detected 5′-exonuclease assays, and hybridization with PNA and LNA probes, single nucleotide primer extension, and others. However, it will be appreciated that other genetic markers and techniques may be applied in accordance with the invention.
SCID in Pigs
According to one aspect of the invention, the causative mutation of pigs with severe combined immunodeficiency disease (SCID) has been identified. This can be used to identify and select for and create an animal model population wherein the pigs possess a gene having one or more mutations that result in reduced numbers or function of immune effectors. “Immune effectors” refers to cells or products of cells that mediate immune functions. Immune effector cells include monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes. Non-cellular immune effectors include antibodies and immunoglobulins or fragments thereof, cytokines, chemokines, and complement. In a more preferred embodiment, the mutations of the invention result in reduced presence or activity of T-lymphocytes, B-lymphocytes, and/or antibodies and immunoglobulins or fragments thereof.
In a preferred embodiment, the mutations are in the Artemis gene, resulting in reduced expression or function of the Artemis protein gene product. The mutations may be one or more of an insertion, a deletion, an inversion, or a single nucleotide polymorphism in the Artemis gene, including, for example, one ore more single nucleotide polymorphisms including an a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45.
In one aspect of the invention, the mutations of the present invention result in loss of expression of the gene product encoded by the Artemis gene, or expression of a truncated gene product encoded by the Artemis gene. The loss of expression or truncation may be due to alterations at one or more splice sites in the Artemis gene.
In another aspect of the invention, pigs are provided that have severe combined immunodeficiency disease (SCID). Such SCID-affected pigs may have or more of an insertion, a deletion, an inversion, or a single nucleotide polymorphism in the Artemis gene, including a G to A substitution at position 25,487 or a G to A substuitution at position 31,214, as determined by reference to SEQ ID NO:45.
In another aspect of the invention, non-naturally occurring nucleotide sequences encoding Artemis protein are provided. In one embodiment, the nucleotide sequence may be provided to an individual affected by SCID, for example by introducing a vector comprising the nucleotide sequence operatively linked to a heterologous promoter sequence. In one embodiment, the nucleotide sequence is SEQ ID NO:5, or encodes a polynucleotide of SEQ ID NO:6.
In another aspect of the invention, pigs are provided with SCID comprising at least one heterologous gene encoding a disruption of the endogenous Artemis gene of said animal, wherein said disruption inactivates said gene.
In a further aspect, the invention provides porcine semen comprising sperm cells from a porcine subject with SCID, or a genetically modified primary cell or embryo, comprising one or more of an insertion, a deletion, an inversion, or a single nucleotide polymorphism in the Artemis gene of said animal, including one or more of G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45. In a further embodiment, the invention provides a pig or pigs produced from the sperm, cell or embryo.
Genetic Tests
The invention includes the discovery of the causative mutation for SCID in pigs. This can be used to develop genetic tests to screen for the mutation or mutations in genetic disequilibrium to the same to identify the presence of SCID in a biological sample from a pig. This allows one to create test populations of pigs to test for agents which ameliorate the disease, or to identify and treat those subject susceptible to the same, or to cull subjects from a breeding population based upon early detection. The method according to the invention includes the provision of a sample of porcine genetic material. Such porcine genetic (DNA) material may be provided by any conventional method or means. The porcine DNA material may for example be extracted, isolated, and purified from blood (e.g., fresh or frozen), tissue samples (e.g., spleen, buccal smears), hair samples containing follicular cells, or semen.
As previously described, the method of the present invention further comprises a step of detecting in the genetic material the presence or absence of a genetic marker that is linked to or preferably is the causative mutation for SCID. In a more preferred embodiment, the invention provides methods for detecting mutations onto chromosome 10 associated with the SCID phenotype. In an even more preferred embodiment, the invention provides methods for detecting mutations to the Artemis gene, including one or more of a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45. In another embodiment, the invention provides methods for detecting one or more SNPs in the 5′ and 3′ regions flanking the Artemis gene, including an A→C substitution at position 51153137 of chromosome 10 (reverse complement of nucleotides at position 20 of SEQ ID NO:12 in the region amplified by the primer pairs of SEQ ID NOS: 11 and 12; and position 19 of SEQ ID NO:14 in the region amplified by the primer pairs of SEQ ID NOS:13 and 14); a G→A substitution at position 51812252 of chromosome 10 (nucleotide at position 25 of SEQ ID NO:17 in the region amplified by the primer pairs of SEQ ID NOS: 16 and 17, and, reverse complement of nucleotide at position 26 of SEQ ID NO:16, respectively, in the region amplified by the primer pairs of SEQ ID NOS: 15 and 16); an A→G substitution at position 51975024 of chromosome 10 (reverse complement of nucleotide at position 19 of SEQ ID NO:20 in the region amplified by the primer pairs of SEQ ID NOS:19 and 20; and nucleotide at position 18 of SEQ ID NO:21, in the region amplified by the primer pairs of SEQ ID NOS:21 and 22); an A→C substitution at position 52066694 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:24 in the region amplified by the primer pairs of SEQ ID NOS:23 and 24; and reverse complement of nucleotide at position 23 of SEQ ID NO: 26 in the region amplified by the primer pairs of SEQ ID NOS:25 and 26); a G→A substitution at position 52086867 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:28 in the region amplified by the primer pairs of SEQ ID NOS:27 and 28; and reverse complement of nucleotide at position 23 of SEQ ID NO:30 in the region amplified by the primer pairs of SEQ ID NOS:29 and 30); A→C substitution at position 52109172 of chromosome 10 (reverse complement of nucleotide at position 18 of SEQ ID NO:32 in the region amplified by the primer pairs of SEQ ID NOS:31 and 32; and reverse complement of nucleotide at position 18 of SEQ ID NO: 34 in the region amplified by the primer pairs of SEQ ID NOS:33 and 34); and/or G→A substitution at position 52174549 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:36 in the region amplified by the primer pairs of SEQ ID NOS:35 and 36; and reverse complement of nucleotide at position 23 of SEQ ID NO:38 in the region amplified by the primer pairs of SEQ ID NOS:37 and 38).
Subjects that have a single allele with mutations to the Artemis gene, and are therefore heterozygous for a SCID mutation, are referred to as “SCID-carrier” subjects. Subjects that have two alleles with mutations to the Artemis gene resulting in decreased expression or activity of the Artemis protein gene product, are referred to as “SCID-affected” subjects or as “SCID pig(s)”. A SCID-affected subject may have two alleles with the same mutation or mutations, or two alleles with different mutation or mutations.
In order to detect if the genetic marker is present in the genetic material, standard methods well known to persons skilled in the art may be applied, for example by the use of nucleic acid amplification. In order to determine if the genetic marker is genetically linked to SCID, a lod score can be applied. A lod score, which is also sometimes referred to as Zmax, indicates the probability (the logarithm of the ratio of the likelihood) that a genetic marker locus and a specific gene locus are linked at a particular distance. Lod scores may be calculated by applying a computer program such as the MLINK program of the LINKAGE package (Lathrop et al., 1985). A lod score of greater than 3.0 is considered to be significant evidence for linkage between the genetic marker and the SCID trait or gene locus.
In one aspect of the invention, determining whether a pig is a SCID-affected or SCID-carrier may be carried out by obtaining a sample of genetic material from one or more pigs, and determining whether one or more mutations are present that cause reduced expression or activity of the Artmeis protein. The method may further include correlating whether an association exists between said allele and SCID in the pig. In a further aspect of the invention, the method may comprise detection of one or more of a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45, or one or more SNPs in the 5′ and 3′ regions flanking the Artemis gene, including an A→C substitution at position 51153137 of chromosome 10 (reverse complement of nucleotides at position 20 of SEQ ID NO:12 in the region amplified by the primer pairs of SEQ ID NOS: 11 and 12; and position 19 of SEQ ID NO:14 in the region amplified by the primer pairs of SEQ ID NOS:13 and 14); a G→A substitution at position 51812252 of chromosome 10 (nucleotide at position 25 of SEQ ID NO:17 in the region amplified by the primer pairs of SEQ ID NOS: 16 and 17, and, reverse complement of nucleotide at position 26 of SEQ ID NO:16, respectively, in the region amplified by the primer pairs of SEQ ID NOS: 15 and 16); an A→G substitution at position 51975024 of chromosome 10 (reverse complement of nucleotide at position 19 of SEQ ID NO:20 in the region amplified by the primer pairs of SEQ ID NOS:19 and 20; and nucleotide at position 18 of SEQ ID NO:21, in the region amplified by the primer pairs of SEQ ID NOS:21 and 22); an A→C substitution at position 52066694 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:24 in the region amplified by the primer pairs of SEQ ID NOS:23 and 24; and reverse complement of nucleotide at position 23 of SEQ ID NO: 26 in the region amplified by the primer pairs of SEQ ID NOS:25 and 26); a G→A substitution at position 52086867 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:28 in the region amplified by the primer pairs of SEQ ID NOS:27 and 28; and reverse complement of nucleotide at position 23 of SEQ ID NO:30 in the region amplified by the primer pairs of SEQ ID NOS:29 and 30); A→C substitution at position 52109172 of chromosome 10 (reverse complement of nucleotide at position 18 of SEQ ID NO:32 in the region amplified by the primer pairs of SEQ ID NOS:31 and 32; and reverse complement of nucleotide at position 18 of SEQ ID NO: 34 in the region amplified by the primer pairs of SEQ ID NOS:33 and 34); and/or G→A substitution at position 52174549 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:36 in the region amplified by the primer pairs of SEQ ID NOS:35 and 36; and reverse complement of nucleotide at position 23 of SEQ ID NO:38 in the region amplified by the primer pairs of SEQ ID NOS:37 and 38). In an even more preferred embodiment, such detection is carried out using one or more of the primer pairs disclosed in Table 1, below.
In another aspect of the invention, detection of a mutation or mutations of the invention is carried out using oligonucleotides that hybridizes to either a normal or a mutant Artemis gene that is capable of detecting one or more mutations selected from the group consisting of a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45., or one or more SNPs in the 5′ and 3′ regions flanking the Artemis gene, including an A→C substitution at position 51153137 of chromosome 10 (reverse complement of nucleotides at position 20 of SEQ ID NO:12 in the region amplified by the primer pairs of SEQ ID NOS: 11 and 12; and position 19 of SEQ ID NO:14 in the region amplified by the primer pairs of SEQ ID NOS:13 and 14); a G→A substitution at position 51812252 of chromosome 10 (nucleotide at position 25 of SEQ ID NO:17 in the region amplified by the primer pairs of SEQ ID NOS: 16 and 17, and, reverse complement of nucleotide at position 26 of SEQ ID NO:16, respectively, in the region amplified by the primer pairs of SEQ ID NOS: 15 and 16); an A→G substitution at position 51975024 of chromosome 10 (reverse complement of nucleotide at position 19 of SEQ ID NO:20 in the region amplified by the primer pairs of SEQ ID NOS:19 and 20; and nucleotide at position 18 of SEQ ID NO:21, in the region amplified by the primer pairs of SEQ ID NOS:21 and 22); an A→C substitution at position 52066694 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:24 in the region amplified by the primer pairs of SEQ ID NOS:23 and 24; and reverse complement of nucleotide at position 23 of SEQ ID NO: 26 in the region amplified by the primer pairs of SEQ ID NOS:25 and 26); a G→A substitution at position 52086867 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:28 in the region amplified by the primer pairs of SEQ ID NOS:27 and 28; and reverse complement of nucleotide at position 23 of SEQ ID NO:30 in the region amplified by the primer pairs of SEQ ID NOS:29 and 30); A→C substitution at position 52109172 of chromosome 10 (reverse complement of nucleotide at position 18 of SEQ ID NO:32 in the region amplified by the primer pairs of SEQ ID NOS:31 and 32; and reverse complement of nucleotide at position 18 of SEQ ID NO: 34 in the region amplified by the primer pairs of SEQ ID NOS:33 and 34); and/or G→A substitution at position 52174549 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:36 in the region amplified by the primer pairs of SEQ ID NOS:35 and 36; and reverse complement of nucleotide at position 23 of SEQ ID NO:38 in the region amplified by the primer pairs of SEQ ID NOS:37 and 38).
It will be appreciated that, in order to detect the specific allele present in a porcine subject with SCID, more than one genetic marker may be applied in accordance with the invention. Thus, at least one marker can be a combination of two or more genetic markers that are shown to be informative whereby the accuracy of the test can be increased.
Genetic markers of the present invention can be made using different methodologies known to those skilled in the art. Thus, it will be understood that, with the knowledge presented herein and the nucleotide sequences of the porcine Artemis gene, which are known and publically available, that additional markers in this gene may be identified and used according to the invention.
Genotyping is based on the analysis of genomic DNA that can be provided by using standard DNA extraction methods as described herein. When the genomic DNA is isolated and purified, nucleic acid amplification (e.g. polymerase chain reaction) can be used to amplify the region of the DNA corresponding to each genetic marker to be used in the analysis for detecting the presence in a porcine subject of a genetic marker associated with SCID.
In another embodiment, the invention comprises a method for identifying genetic markers for SCID in general. Once a major effect gene has been identified, it is expected that other variations present in the same gene, allele or in sequences in useful linkage disequilibrium therewith, may be used to identify similar effects on these traits without undue experimentation. The identification of other such genetic variation, once a major effect gene has been discovered, represents no more than routine screening and optimization of parameters well known to those of skilled in the art and is intended to be within the scope of this invention. This can include other lines, breeds, or even other meat animals.
The present invention provides a method of genotyping an animal comprising a polynucleotide of the present invention. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in an animal population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among animal species, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. The particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques described hereinafter.
In the present invention, the nucleic acid probes may be employed for molecular marker mapping of nuclear genomes which hybridize, under selective hybridization conditions, to the variant polynucleotide of the present invention. In preferred embodiments, the probes are selected from polynucleotides of the present invention. Typically, these probes are cDNA probes or restriction enzyme treated (e.g., PST I) genomic clones. The length of the probes is typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length. Preferably, the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement.
Methods for detecting or differentiating polymorphic or allelic variants of polynucleotides would be known to one of ordinary skill in the art, including restriction fragment length polymorphism (RFLP); single stranded conformation analysis (SSCA); denaturing gradient gel electrophoresis (DGGE); RNase protection assays; allele-specific oligonucleotides (ASOs); the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and allele-specific PCR. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage (CMC). Thus, the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe. The nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample. In preferred embodiments, the nucleic acid probe comprises a polynucleotide of the present invention.
The following is a general overview of techniques that can be used to assay for the polymorphisms of the invention.
In the present invention, a sample of genetic material is obtained from an animal. Samples can be obtained from blood, tissue, semen, etc. Generally, peripheral blood cells are used as the source, and the genetic material is DNA. A sufficient amount of cells are obtained to provide a sufficient amount of DNA for analysis. This amount will be known or readily determinable by those skilled in the art. The DNA is isolated from the blood cells by techniques known to those skilled in the art.
Artemis Protein and Artemis-Encoding Polynucleotides
In one aspect of the invention, novel polynucleotide sequences are provided. These sequences relate to the Artemis gene, and include novel mutations that result in, or are related to, a loss of expression or function of Artemis protein causing SCID. These sequences include non-naturally occurring cDNA sequences, as well as novel sequences encompassing non-coding regions of the Artemis gene and surrounding untranslated regions (UTRs). In one embodiment, the sequences include, for example, SEQ ID NOS:1, 3, 5, and 7-10. In another embodiment, the sequences include the regions amplified by the primer pairs provided in Table 1 (SEQ ID NOS:11-38).
In another aspect, the invention encompasses these novel sequences within polynucleotide constructs such as, for example, vectors. In one embodiment, these constructs may be used to generate cells or animals with reduced Artemis expression or activity. In another aspect, these constructs may be used to enhance Artemis expression or activity, or to correct an existing defect in the expression or activity of endogenous Artemis in a cell or organism. In one embodiment, the constructs may be provided as a pharmaceutical composition for the treatment of SCID.
Isolation and Amplification of Nucleic Acid
Samples of genomic DNA are isolated from any convenient source, including saliva, buccal cells, hair roots, blood, cord blood, amniotic fluid, interstitial fluid, peritoneal fluid, chorionic villus, and any other suitable cell or tissue sample with intact interphase nuclei or metaphase cells. The cells can be obtained from solid tissue, as from a fresh or preserved organ, or from a tissue sample or biopsy. The sample can contain compounds that are not naturally intermixed with the biological material, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
Methods for isolation of genomic DNA from these various sources are described in, for example, Kirby, DNA Fingerprinting, An Introduction, W.H. Freeman & Co. New York (1992). Genomic DNA can also be isolated from cultured primary or secondary cell cultures or from transformed cell lines derived from any of the aforementioned tissue samples.
Samples of animal RNA can also be used. RNA can be isolated from tissues expressing the major effect gene of the invention, as described in Sambrook et al., supra. RNA can be total cellular RNA, mRNA, poly A+RNA, or any combination thereof. For best results, the RNA is purified, but can also be unpurified cytoplasmic RNA. RNA can be reverse transcribed to form DNA, which is then used as the amplification template, so that the PCR indirectly amplifies a specific population of RNA transcripts. See, e.g., Sambrook, supra, Kawasaki et al., Chapter 8 in PCR Technology, (1992) supra, and Berg et al., Hum. Genet. 85:655-658 (1990).
In one aspect, nucleic acid may be obtained and amplified to determine the presence or absence of one or more mutations associated with SCID in a pig. In a preferred embodiment, nucleic acid may be obtained and amplified to detect one or more of a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45, or one or more SNPs in the 5′ and 3′ regions flanking the Artemis gene, including an A→C substitution at position 51153137 of chromosome 10 (reverse complement of nucleotides at position 20 of SEQ ID NO:12 in the region amplified by the primer pairs of SEQ ID NOS: 11 and 12; and position 19 of SEQ ID NO:14 in the region amplified by the primer pairs of SEQ ID NOS:13 and 14); a G→A substitution at position 51812252 of chromosome 10 (nucleotide at position 25 of SEQ ID NO:17 in the region amplified by the primer pairs of SEQ ID NOS: 16 and 17, and, reverse complement of nucleotide at position 26 of SEQ ID NO:16, respectively, in the region amplified by the primer pairs of SEQ ID NOS: 15 and 16); an A→G substitution at position 51975024 of chromosome 10 (reverse complement of nucleotide at position 19 of SEQ ID NO:20 in the region amplified by the primer pairs of SEQ ID NOS:19 and 20; and nucleotide at position 18 of SEQ ID NO:21, in the region amplified by the primer pairs of SEQ ID NOS:21 and 22); an A→C substitution at position 52066694 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:24 in the region amplified by the primer pairs of SEQ ID NOS:23 and 24; and reverse complement of nucleotide at position 23 of SEQ ID NO: 26 in the region amplified by the primer pairs of SEQ ID NOS:25 and 26); a G→A substitution at position 52086867 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:28 in the region amplified by the primer pairs of SEQ ID NOS:27 and 28; and reverse complement of nucleotide at position 23 of SEQ ID NO:30 in the region amplified by the primer pairs of SEQ ID NOS:29 and 30); A→C substitution at position 52109172 of chromosome 10 (reverse complement of nucleotide at position 18 of SEQ ID NO:32 in the region amplified by the primer pairs of SEQ ID NOS:31 and 32; and reverse complement of nucleotide at position 18 of SEQ ID NO: 34 in the region amplified by the primer pairs of SEQ ID NOS:33 and 34); and/or G→A substitution at position 52174549 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:36 in the region amplified by the primer pairs of SEQ ID NOS:35 and 36; and reverse complement of nucleotide at position 23 of SEQ ID NO:38 in the region amplified by the primer pairs of SEQ ID NOS:37 and 38). In an even more preferred embodiment, such detection is carried out using one or more of the primer pairs disclosed in Table 1, below.
PCR Amplification
The most common means for amplification is polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,965,188 each of which is hereby incorporated by reference. Without limitation, PCR may be used to amplify genetic material obtained from blood cells, tissues, or other physiological sources. One of skill in the art would understand that if PCR is used to amplify the target regions in blood cells, for example, the blood can be fresh or frozen, heparinized, or untreated, or otherwise processed. Cells in other physiological fluids may also be assayed.
One of skill in the art would understand how to process fresh or preserved tissues. For example, and without limitation, tissues may be roughly minced using a sterile, disposable scalpel and a sterile needle (or two scalpels) in a 5 mm Petri dish. Procedures for removing paraffin from tissue sections are described in a variety of specialized handbooks well known to those skilled in the art.
To amplify a target nucleic acid sequence in a sample by PCR, the sequence must be accessible to the components of the amplification system. One of ordinary skill in the art would understand various methods for isolating target DNA. For example, one method useful for isolating target DNA from relatively large samples is crude extraction. Briefly, mononuclear cells from samples of blood, amniocyte from amniotic fluid, cultured chorionic villus cells, or the like are isolated by layering on sterile Ficoll-Hypaque gradient by standard procedures. Interphase cells are collected and washed three times in sterile phosphate buffered saline before DNA extraction. The cells are resuspended (106 nucleated cells per 100 μl) in a buffer of 50 mM Tris-HCl (pH 8.3), 50 mM KCl 1.5 mM MgCl2, 0.5% Tween 20, 0.5% NP40 supplemented with 100 μg/ml of proteinase K. After incubating at 56° C. for 2 hr. the cells are heated to 95° C. for 10 min to inactivate the proteinase K and immediately moved to wet ice (snap-cool). If gross aggregates are present, another cycle of digestion in the same buffer should be undertaken. Ten μl of this extract is used for amplification.
One of ordinary skill in the art would understand methods for extracting DNA from tissues. For example, and without limitation, when extracting DNA from tissues, such as chorionic villus cells or confluent cultured cells, the amount of the above mentioned buffer with proteinase K may vary according to the size of the tissue sample. The extract is incubated for 4-10 hrs at 50°-60° C. and then at 95° C. for 10 minutes to inactivate the proteinase. During longer incubations, fresh proteinase K should be added after about 4 hr at the original concentration.
When the sample contains a small number of cells, extraction may be accomplished by methods as described in Higuchi, “Simple and Rapid Preparation of Samples for PCR”, in PCR Technology, Ehrlich, H. A. (ed.), Stockton Press, New York, which is incorporated herein by reference. PCR can be employed to amplify target regions in very small numbers of cells (1000-5000) derived from individual colonies from bone marrow and peripheral blood cultures. The cells in the sample are suspended in 20 μl of PCR lysis buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20) and frozen until use. When PCR is to be performed, 0.6 μl of proteinase K (2 mg/ml) is added to the cells in the PCR lysis buffer. The sample is then heated to about 60° C. and incubated for 1 hr. Digestion is stopped through inactivation of the proteinase K by heating the samples to 95° C. for 10 min and then cooling on ice.
A relatively easy procedure for extracting DNA for PCR is a salting out procedure adapted from the method described by Miller et al., Nucleic Acids Res. 16:1215 (1988), which is incorporated herein by reference. Mononuclear cells are separated on a Ficoll-Hypaque gradient. The cells are resuspended in 3 ml of lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM Na2 EDTA, pH 8.2). Fifty μl of a 20 mg/ml solution of proteinase K and 150 μl of a 20% SDS solution are added to the cells and then incubated at 37° C. overnight. Rocking the tubes during incubation will improve the digestion of the sample. If the proteinase K digestion is incomplete after overnight incubation (fragments are still visible), an additional 50 μl of the 20 mg/ml proteinase K solution is mixed in the solution and incubated for another night at 37° C. on a gently rocking or rotating platform. Following adequate digestion, one ml of a 6 M NaCl solution is added to the sample and vigorously mixed. The resulting solution is centrifuged for 15 minutes at 3000 rpm. The pellet contains the precipitated cellular proteins, while the supernatant contains the DNA. The supernatant is removed to a 15 ml tube that contains 4 ml of isopropanol. The contents of the tube are mixed gently until the water and the alcohol phases have mixed and a white DNA precipitate has formed. The DNA precipitate is removed and dipped in a solution of 70% ethanol and gently mixed. The DNA precipitate is removed from the ethanol and air-dried. The precipitate is placed in distilled water and dissolved.
Kits for the extraction of high-molecular weight DNA for PCR include a Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, Ind.), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, Md.), Elu-Quik DNA Purification Kit (Schleicher & Schuell, Keene, N.H.), DNA Extraction Kit (Stratagene, LaJolla, Calif.), TurboGen Isolation Kit (Invitrogen, San Diego, Calif.), DNeasy Blood & Tissue Kit (Qiagen Inc, Valencia, Calif.), and the like. Use of these kits according to the manufacturer's instructions is generally acceptable for purification of DNA prior to practicing the methods of the present invention.
The concentration and purity of the extracted DNA can be determined by spectrophotometric analysis of the absorbance of a diluted aliquot at 260 nm and 280 nm. After extraction of the DNA, PCR amplification may proceed. The first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
In a particularly useful embodiment of PCR amplification, strand separation is achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase (see U.S. Pat. No. 4,965,188, incorporated herein by reference). Typical heat denaturation involves temperatures ranging from about 80° C. to 105° C. for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity. For example, the enzyme RecA has helicase activity in the presence of ATP. The reaction conditions suitable for strand separation by helicases are known in the art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, each of which is incorporated herein by reference).
Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering systems. Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis. In some cases, the target regions may encode at least a portion of a protein expressed by the cell. In this instance, mRNA may be used for amplification of the target region. Alternatively, PCR can be used to generate a cDNA library from RNA for further amplification, the initial template for primer extension is RNA. Polymerizing agents suitable for synthesizing a complementary, copy-DNA (cDNA) sequence from the RNA template are reverse transcriptase (RT), such as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, or Therms thermophilus (Tth) DNA polymerase, a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer Cetus, Inc. Typically, the genomic RNA template is heat degraded during the first denaturation step after the initial reverse transcription step leaving only DNA template. Suitable polymerases for use with a DNA template include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus and commercially available from Perkin Elmer Cetus, Inc. The latter enzyme is widely used in the amplification and sequencing of nucleic acids. The reaction conditions for using Taq polymerase are known in the art and are described in Gelfand, 1989, PCR Technology, supra.
Allele Specific PCR
Allele-specific PCR differentiates between target regions differing in the presence of absence of a variation or polymorphism. PCR amplification primers are chosen that bind only to certain alleles of the target sequence. This method is described by Gibbs, Nucleic Acid Res. 17:12427-2448 (1989).
Allele Specific Oligonucleotide Screening Methods
Further diagnostic screening methods employ the allele-specific oligonucleotide (ASO) screening methods, as described by Saiki et al., Nature 324:163-166 (1986). Oligonucleotides with one or more base pair mismatches are generated for any particular allele. ASO screening methods detect mismatches between variant target genomic or PCR amplified DNA and non-mutant oligonucleotides, showing decreased binding of the oligonucleotide relative to a mutant oligonucleotide. Oligonucleotide probes can be designed that under low stringency they will bind to both polymorphic forms of the allele, but at high stringency, bind to the allele to which they correspond. Alternatively, stringency conditions can be devised in which an essentially binary response is obtained—an ASO corresponding to a variant form of the target gene will hybridize to that allele, and not to the wild type allele.
Ligase Mediated Allele Detection Method
Target regions of a test subject's DNA can be compared with target regions in unaffected and affected family members by ligase-mediated allele detection. See Landegren et al., Science 241:107-1080 (1988). Ligase may also be used to detect point mutations in the ligation amplification reaction described in Wu et al., Genomics 4:560-569 (1989). The ligation amplification reaction (LAR) utilizes amplification of specific DNA sequence using sequential rounds of template dependent ligation as described in Wu, supra, and Barany, Proc. Nat. Acad. Sci. 88:189-193 (1990).
Denaturing Gradient Gel Electrophoresis
Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. DNA molecules melt in segments, termed melting domains, under conditions of increased temperature or denaturation. Each melting domain melts cooperatively at a distinct, base-specific melting temperature (Tm). Melting domains are at least 20 base pairs in length, and may be up to several hundred base pairs in length.
Differentiation between alleles based on sequence specific melting domain differences can be assessed using polyacrylamide gel electrophoresis, as described in Chapter 7 of Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, W.H. Freeman and Co., New York (1992), the contents of which are hereby incorporated by reference.
Generally, a target region to be analyzed by denaturing gradient gel electrophoresis is amplified using PCR primers flanking the target region. The amplified PCR product is applied to a polyacrylamide gel with a linear denaturing gradient as described in Myers et al., Meth. Enzymol. 155:501-527 (1986), and Myers et al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95-139 (1988), the contents of which are hereby incorporated by reference. The electrophoresis system is maintained at a temperature slightly below the Tm of the melting domains of the target sequences.
In an alternative method of denaturing gradient gel electrophoresis, the target sequences may be initially attached to a stretch of GC nucleotides, termed a GC clamp, as described in Chapter 7 of Erlich, supra. Preferably, at least 80% of the nucleotides in the GC clamp are either guanine or cytosine. Preferably, the GC clamp is at least 30 bases long. This method is particularly suited to target sequences with high Tm's.
Generally, the target region is amplified by the polymerase chain reaction as described above. One of the oligonucleotide PCR primers carries at its 5′ end, the GC clamp region, at least 30 bases of the GC rich sequence, which is incorporated into the 5′ end of the target region during amplification. The resulting amplified target region is run on an electrophoresis gel under denaturing gradient conditions as described above. DNA fragments differing by a single base change will migrate through the gel to different positions, which may be visualized by ethidium bromide staining.
Temperature Gradient Gel Electrophoresis
Temperature gradient gel electrophoresis (TGGE) is based on the same underlying principles as denaturing gradient gel electrophoresis, except the denaturing gradient is produced by differences in temperature instead of differences in the concentration of a chemical denaturant. Standard TGGE utilizes an electrophoresis apparatus with a temperature gradient running along the electrophoresis path. As samples migrate through a gel with a uniform concentration of a chemical denaturant, they encounter increasing temperatures. An alternative method of TGGE, temporal temperature gradient gel electrophoresis (TTGE or tTGGE) uses a steadily increasing temperature of the entire electrophoresis gel to achieve the same result. As the samples migrate through the gel the temperature of the entire gel increases, leading the samples to encounter increasing temperature as they migrate through the gel. Preparation of samples, including PCR amplification with incorporation of a GC clamp, and visualization of products are the same as for denaturing gradient gel electrophoresis.
Single-Strand Conformation Polymorphism Analysis
Target sequences or alleles at an particular locus can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 85:2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. Thus, electrophoretic mobility of single-stranded amplification products can detect base-sequence difference between alleles or target sequences.
Chemical or Enzymatic Cleavage of Mismatches
Differences between target sequences can also be detected by differential chemical cleavage of mismatched base pairs, as described in Grompe et al., Am. J. Hum. Genet. 48:212-222 (1991). In another method, differences between target sequences can be detected by enzymatic cleavage of mismatched base pairs, as described in Nelson et al., Nature Genetics 4:11-18 (1993). Briefly, genetic material from an animal and an affected family member may be used to generate mismatch free heterohybrid DNA duplexes. As used herein, “heterohybrid” means a DNA duplex strand comprising one strand of DNA from one animal, and a second DNA strand from another animal, usually an animal differing in the phenotype for the trait of interest. Positive selection for heterohybrids free of mismatches allows determination of small insertions, deletions or other polymorphisms that may be associated with polymorphisms.
Non-Gel Systems
Other possible techniques include non-gel systems such as TaqManTM (Perkin Elmer). In this system oligonucleotide PCR primers are designed that flank the mutation in question and allow PCR amplification of the region. A third oligonucleotide probe is then designed to hybridize to the region containing the base subject to change between different alleles of the gene. This probe is labeled with fluorescent dyes at both the 5′ and 3′ ends. These dyes are chosen such that while in this proximity to each other the fluorescence of one of them is quenched by the other and cannot be detected. Extension by Taq DNA polymerase from the PCR primer positioned 5′ on the template relative to the probe leads to the cleavage of the dye attached to the 5′ end of the annealed probe through the 5′ nuclease activity of the Taq DNA polymerase. This removes the quenching effect allowing detection of the fluorescence from the dye at the 3′ end of the probe. The discrimination between different DNA sequences arises through the fact that if the hybridization of the probe to the template molecule is not complete—i.e. there is a mismatch of some form—the cleavage of the dye does not take place. Thus, only if the nucleotide sequence of the oligonucleotide probe is completely complimentary to the template molecule to which it is bound will quenching be removed. A reaction mix can contain two different probe sequences each designed against different alleles that might be present thus allowing the detection of both alleles in one reaction.
Yet another technique includes an Invader Assay which includes isothermic amplification that relies on a catalytic release of fluorescence. See Third Wave Technology
Non-PCR Based DNA Diagnostics
The identification of a DNA sequence linked to an allele sequence can be made without an amplification step, based on polymorphisms including restriction fragment length polymorphisms in an animal and a family member. Hybridization probes are generally oligonucleotides which bind through complementary base pairing to all or part of a target nucleic acid. Probes typically bind target sequences lacking complete complementarity with the probe sequence depending on the stringency of the hybridization conditions. The probes are preferably labeled directly or indirectly, such that by assaying for the presence or absence of the probe, one can detect the presence or absence of the target sequence. Direct labeling methods include radioisotope labeling, such as with 32P or 35S. Indirect labeling methods include fluorescent tags, biotin complexes which may be bound to avidin or streptavidin, or peptide or protein tags. Visual detection methods include photoluminescents, Texas red, rhodamine and its derivatives, red leuco dye and 3,3′,5,5′-tetramethylbenzidine (TMB), fluorescein, and its derivatives, dansyl, umbelliferone and the like or with horse radish peroxidase, alkaline phosphatase and the like.
Hybridization probes include any nucleotide sequence capable of hybridizing to a porcine chromosome where one of the major effect genes resides, and thus defining a genetic marker linked to one of the major effect genes, including a restriction fragment length polymorphism, a hypervariable region, repetitive element, or a variable number tandem repeat. Hybridization probes can be any gene or a suitable analog. Further suitable hybridization probes include exon fragments or portions of cDNAs or genes known to map to the relevant region of the chromosome.
Preferred tandem repeat hybridization probes for use according to the present invention are those that recognize a small number of fragments at a specific locus at high stringency hybridization conditions, or that recognize a larger number of fragments at that locus when the stringency conditions are lowered.
One or more additional restriction enzymes and/or probes and/or primers can be used. Additional enzymes, constructed probes, and primers can be determined by routine experimentation by those of ordinary skill in the art and are intended to be within the scope of the invention.
Although the methods described herein may be in terms of the use of a single restriction enzyme and a single set of primers, the methods are not so limited. One or more additional restriction enzymes and/or probes and/or primers can be used, if desired. Indeed in some situations it may be preferable to use combinations of markers giving specific haplotypes. Additional enzymes, constructed probes and primers can be determined through routine experimentation, combined with the teachings provided and incorporated herein.
According to one embodiment of the invention, polymorphisms in a major effect gene cause SCID. The presence or absence of the markers, in one embodiment may be assayed by PCR RFLP analysis using if needed, restriction endonucleases, and amplification primers which may be designed using analogous human, pig or other of the sequences due to the high homology in the region surrounding the polymorphisms, or may be designed using known sequences (for example, human) as exemplified in GenBank or even designed from sequences obtained from linkage data from closely surrounding genes based upon the teachings and references herein. The sequences surrounding the polymorphism will facilitate the development of alternate PCR tests in which a primer of about 4-30 contiguous bases taken from the sequence immediately adjacent to the polymorphism is used in connection with a polymerase chain reaction to greatly amplify the region before treatment with the desired restriction enzyme. The primers need not be the exact complement; substantially equivalent sequences are acceptable. The design of primers for amplification by PCR is known to those of skill in the art and is discussed in detail in Ausubel (ed.), Short Protocols in Molecular Biology, Fourth Edition, John Wiley and Sons 1999. The following is a brief description of primer design.
In one embodiment, such non-PCR based DNA diagnostic— methods may be used to detect one or more of a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45, or one or more SNPs in the 5′ and 3′ regions flanking the Artemis gene, including an A→C substitution at position 51153137 of chromosome 10 (reverse complement of nucleotides at position 20 of SEQ ID NO:12 in the region amplified by the primer pairs of SEQ ID NOS: 11 and 12; and position 19 of SEQ ID NO:14 in the region amplified by the primer pairs of SEQ ID NOS:13 and 14); a G→A substitution at position 51812252 of chromosome 10 (nucleotide at position 25 of SEQ ID NO:17 in the region amplified by the primer pairs of SEQ ID NOS: 16 and 17, and, reverse complement of nucleotide at position 26 of SEQ ID NO:16, respectively, in the region amplified by the primer pairs of SEQ ID NOS: 15 and 16); an A→G substitution at position 51975024 of chromosome 10 (reverse complement of nucleotide at position 19 of SEQ ID NO:20 in the region amplified by the primer pairs of SEQ ID NOS:19 and 20; and nucleotide at position 18 of SEQ ID NO:21, in the region amplified by the primer pairs of SEQ ID NOS:21 and 22); an A→C substitution at position 52066694 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:24 in the region amplified by the primer pairs of SEQ ID NOS:23 and 24; and reverse complement of nucleotide at position 23 of SEQ ID NO: 26 in the region amplified by the primer pairs of SEQ ID NOS:25 and 26); a G→A substitution at position 52086867 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:28 in the region amplified by the primer pairs of SEQ ID NOS:27 and 28; and reverse complement of nucleotide at position 23 of SEQ ID NO:30 in the region amplified by the primer pairs of SEQ ID NOS:29 and 30); A→C substitution at position 52109172 of chromosome 10 (reverse complement of nucleotide at position 18 of SEQ ID NO:32 in the region amplified by the primer pairs of SEQ ID NOS:31 and 32; and reverse complement of nucleotide at position 18 of SEQ ID NO: 34 in the region amplified by the primer pairs of SEQ ID NOS:33 and 34); and/or G→A substitution at position 52174549 of chromosome 10 (reverse complement of nucleotide at position 23 of SEQ ID NO:36 in the region amplified by the primer pairs of SEQ ID NOS:35 and 36; and reverse complement of nucleotide at position 23 of SEQ ID NO:38 in the region amplified by the primer pairs of SEQ ID NOS:37 and 38).
Primer Design Strategy
Increased use of polymerase chain reaction (PCR) methods has stimulated the development of many programs to aid in the design or selection of oligonucleotides used as primers for PCR. Four examples of such programs that are freely available via the Internet are: PRIMER by Mark Daly and Steve Lincoln of the Whitehead Institute (UNIX, VMS, DOS, and Macintosh), Oligonucleotide Selection Program (OSP) by Phil Green and LaDeana Hiller of Washington University in St. Louis (UNIX, VMS, DOS, and Macintosh), PGEN by Yoshi (DOS only), and Amplify by Bill Engels of the University of Wisconsin (Macintosh only). Generally these programs help in the design of PCR primers by searching for bits of known repeated-sequence elements and then optimizing the Tm by analyzing the length and GC content of a putative primer. Commercial software is also available and primer selection procedures are rapidly being included in most general sequence analysis packages.
Sequencing and PCR Primers
Designing oligonucleotides for use as either sequencing or PCR primers requires selection of an appropriate sequence that specifically recognizes the target, and then testing the sequence to eliminate the possibility that the oligonucleotide will have a stable secondary structure. Inverted repeats in the sequence can be identified using a repeat-identification or RNA-folding program such as those described above (see prediction of Nucleic Acid Structure). If a possible stem structure is observed, the sequence of the primer can be shifted a few nucleotides in either direction to minimize the predicted secondary structure. The sequence of the oligonucleotide should also be compared with the sequences of both strands of the appropriate vector and insert DNA. Obviously, a sequencing primer should only have a single match to the target DNA. It is also advisable to exclude primers that have only a single mismatch with an undesired target DNA sequence. For PCR primers used to amplify genomic DNA, the primer sequence should be compared to the sequences in the GenBank database to determine if any significant matches occur. If the oligonucleotide sequence is present in any known DNA sequence or, more importantly, in any known repetitive elements, the primer sequence should be changed.
The methods and materials of the invention may also be used more generally to evaluate animal DNA, genetically type individual animals, and detect genetic differences in animals. In particular, a sample of animal genomic DNA may be evaluated by reference to one or more controls to determine if a polymorphism in one of the sequences is present. Preferably, RFLP analysis is performed with respect to the animal's sequences, and the results are compared with a control. The control is the result of a RFLP analysis of one or both of the sequences of a different animal where the polymorphism of the animal gene is known. Similarly, the genotype of an animal may be determined by obtaining a sample of its genomic DNA, conducting RFLP analysis of the gene in the DNA, and comparing the results with a control. Again, the control is the result of RFLP analysis of one of the sequences of a different animal. The results genetically type the animal by specifying the polymorphism(s) in its gene. Finally, genetic differences among animals can be detected by obtaining samples of the genomic DNA from at least two animals, identifying the presence or absence of a polymorphism in one of the nucleotide sequences, and comparing the results.
These assays are useful for identifying the genetic markers relating to SCID, as discussed above, for identifying other polymorphisms in the same genes or alleles that may be correlated with other characteristics, and for the general scientific analysis of animal genotypes and phenotypes.
Once a polymorphism has been identified and a correlation to a particular trait established, one of skill in the art will understand that there are many ways to genotype animals for this polymorphism. The design of such alternative tests merely represents optimization of parameters known to those of skill in the art and is intended to be within the scope of this invention as fully described herein. In a preferred embodiment, PCR-based assays use the primers set out in Table 1.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription and Translation (B. D. Hames & S. J. Higgins eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning, (1984).
In one embodiment, a multiplex PCR system is used to detect one or more genetic markers relating to SCID, as discussed above, for identifying other polymorphisms in the same genes or alleles that may be correlated with other characteristics, and for the general scientific analysis of animal genotypes and phenotypes. In a particular embodiment, the multiplex PCR system detects the causative mutation in haplotype 16, found at position 615 of Sequence 4 (SEQ ID NO:7), and/or for the causative mutation in haplotype 12, found at position 116 of Sequence 6 (SEQ ID NO:9). The multiplex PCR system can be any suitable system that can be used to distinguish between nucleotides at one or more locations, such as, for example, the ABI PRISM SNaPshot system.
Creation of Transgenic Animals
Transgenic animals are animals which have had genetic material artificially inserted into the genome. Typically, this material is present as either an extra chromosomal element or is stably integrated into the chromosomal DNA of the organism in at least a subset of cells. Preferably, this includes stable integration into the germline cells. In one aspect, the genetic material comprises a heterologous gene. Typically, a chimeric animal (chimera) is produced, where only a subset of the somatic cells possess the desired transgene. These chimeras are then used for further breeding to generate the ultimate transgenic animal.
A transgenic animal can be a knock-out having a partial or complete loss of function of the target gene. A knock-out of Artemis means that Artemis gene expression is undetectable or insignificant. This can be accomplished through a variety of means including: introduction of a disruption of the coding sequence (e.g. substitutions at splice sites, insertion of one or more stop codons and/or insertion of a DNA fragment), deletion of coding sequence, substitution of stop codons for coding sequence, chromosomal deletion of all or part of the native gene may be induced (including deletions of the non-coding regions, particularly the promoter region, 3′ regulatory sequences, enhancers, or deletions of gene that activate expression of 5-HT6 genes), and/or introduction of an antisense construct that blocks expression of the native genes (for example, see Li and Cohen (1996) Cell 85:319-329). “Knock-outs” also include conditional knockouts, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF). When ES cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting progeny are screened for mutant cells having the construct. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected.
The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture.
In one aspect, these methods are used to produce a SCID pig. In a preferred embodiment, the methods may be used to produce a transgenic pig comprising one or more mutations to the Artemis gene. In a more preferred embodiment, the mutation comprises one or more of a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45. In one embodiment, the transgenic pig comprises a heterologous gene comprising one or more of the mutations. The heterologous gene can be operatively linked to a heterologous promoter sequence and or a terminator sequence.
In another aspect, these methods are used to produce a pig with increased expression of Artermis gene and/or increased activity of Artemis protein. In one aspect, the methods are used to inhibit, treat, or ameliorate a disease or condition. In one embodiment, the invention encompasses a pig comprising a non-naturally occurring nucleotide sequence encoding an Artemis protein operatively linked to a heterologous promoter, wherein the Artemis protein encoded by the nucleotide sequence has nuclease activity.
Increasing the Activity and/or Level of Artemis
Methods are provided to increase the activity and/or level of the Artemis polypeptides to ameliorate the affects caused by loss of function mutations. These methods can be carried out in cell culture. Alternatively, the methods can be carried out in an animal. In one aspect, an increase in the level and/or activity of an artemis polypeptide can be achieved by providing to the animal an artemis polypeptide. The polypeptide can be provided by introducing polypeptide to the animal, introducing to the animal a nucleotide sequence encoding a functional artemis polypeptide, or alternatively by modifying a genomic locus encoding the aberrant artemis polypeptide so that function is restored.
As discussed elsewhere herein, many methods are known in the art of providing a polypeptide to an animal including, but not limited to, direct introduction of the polypeptide to animal, introducing into the animal (transiently or stably) a polynucleotide construct encoding a polypeptide having artemis activity. It is also recognized that the methods of the invention may employ a polynucleotide that is not capable of directing, in the transformed cell, the expression of a protein or an RNA. Thus, the level and/or activity of an artemis polypeptide may be increased by altering the gene encoding the polypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868. Therefore mutagenized animals that carry corrective mutations in the Artemis gene, where the mutations increase expression of a functional gene or increase the activity of the encoded polypeptide are provided.
Reducing the Activity and/or Level of Artemis
Methods are also provided to reduce or eliminate the activity of Artemis by transforming a cell with an expression cassette that expresses a polynucleotide that inhibits the expression of Artemis. These methods can be carried out in cell culture. Alternatively, the methods can be carried out in an animal. The polynucleotide may inhibit the expression of the Artemis directly, by preventing transcription or translation of Artemis messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of an Artemis gene encoding an Artemis. Methods for inhibiting or eliminating the expression of a gene in a cell or animal are well known in the art, and any such method may be used in the present invention to inhibit the expression of the Artemis gene. Many methods may be used to reduce or eliminate the activity of Artemis polypeptide. In addition, more than one method may be used to reduce the activity.
Polypeptide-Based Inhibition of Gene Expression
In one embodiment, the polynucleotide encoding a zinc finger protein that binds to a gene encoding Artemis may be provided, resulting in reduced expression of the gene. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteins to inhibit the expression of genes are described, for example, in U.S. Patent Publication Nos. 2003/0037355; each of which is herein incorporated by reference. In another aspect, a polynucleotide may be provided that encodes a TAL effector protein that targets and disrupts the gene encoding Artemis, resulting in reduced expression of the gene Methods of selecting sites for targeting by TAL effector proteins have been described, and methods for using zinc finger proteins to inhibit the expression of genes are described, for example, in U.S. Pat. No. 8,586,363; which is herein incorporated by reference.
Gene Disruption
In some embodiments of the present invention, the activity of the Artemis protein polypeptide is reduced or eliminated by disrupting the gene encoding the Artemis polypeptide. The gene encoding the Artemis polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing cells using random or targeted mutagenesis, and selecting for cells or animals that have SCID phenotype or reduced Artemis gene product or activity.
Gene Editing
In some embodiments of the present invention, gene editing techniques may be used to alter the nucleotide sequence in the endogenous Artemis gene of pigs. Gene editing may be accomplished using a variety of compositions and methods known in the art, including zinc-finger nucleases (ZFNs), TALENs, the CRISPR/Cas9 system, or equivalent genome editors. Methods for designing and using zinc finger proteins and TALENs are described, for example, in U.S. Pat. Nos. 6,453,242 and 6,534,261, which are incorporated herein in their entirety. In one aspect, the gene editing techniques may be used to introduce a disruption into the Artemis gene, thereby causing SCID in the pig. The disruption may be an insertion, a deletion, an inversion, or a single nucleotide polymorphism in the Artemis gene of said animal. The disruption may be to a coding region, or a non-coding region, including for example, promoter regions, enhancer regions, and/or splice sites. In one embodiment, the gene editing techniques may be used to introduce a G to A substitution at position 25,487 or a G to A substitution at position 31,214, as determined by reference to SEQ ID NO:45. In another aspect gene editing techniques may be used to correct a defect in the Artemis gene in an individual or animal with SCID.
Gene Silencing Using RNA Interference
MicroRNAs (miRNAs) are small non-coding RNAs which negatively regulate protein-coding genes primarily by decreasing stability of RNA transcripts. Decreased stability is typically accomplished through the binding of the miRNA to the target transcript. These binding sites preferentially reside in the 3′ UTRs of the genes, however, they can occur throughout the entire transcript. Generally, miRNA binding sites are well conserved. Some genes which are heavily expressed (e.g. housekeeping genes) have smaller 3′ UTR regions which may reduce the likelihood of binding to miRNAs. Genes with a 3′ UTR longer than 4 kB are less affected by miRNA. Other factors influence the selection and effectiveness of miRNAs. For example, many miRNAs are known to regulate genes involved in cell development processes. Another example is that miRNAs appear to preferentially target genes with high CpG promoters. Also, as highly expressed genes transcribe a large number of mRNAs, the miRNA regulation of those mRNAs can be different from those of weakly expressed genes, although current analyses disagree on whether miRNAs affect highly expressed genes more or less than medium or lowly expressed genes.
Potential miRNAs can be screened using high throughput experiments using microarrays or proteomics. Examples of these high-throughput systems are described in: Lim L P, et al. Nature 2005, 433:769-773; Grimson A, et al., Mol Cell 2007, 27:91-105; Selbach M, Schwanhausser B, et al, Nature 2008, 455:58-63; Baek D, Villén J, et al, Nature 2008, 455:64-71. All of these references are incorporated by reference as if set forth fully herein. Well designed and effective RNA interference will reduce expression by approximately 50%, 60%, 70%, 80%, 90% and/or 100% depending on site selection and other factors listed above. It is well within the skill in the art to design miRNAs which will bind to a target transcript and screen the same for determining effectiveness of the miRNA.
Creation of Microarrays
Microarrays are used to perform large parallel analysis of various biological and chemical samples. A microarray is an array of spots of samples (probes) which are immobilized at specified positions on a substrate. Each spot contains chemical or biological material which is capable of interacting with certain target molecules (such as a DNA sample). In order to use a microarray, it is flooded with a solution containing target molecules. For DNA/RNA applications, the probes are short oligonucleotides which are complimentary to target DNA/RNA samples. When the samples are labeled using fluorescent or radioactive labels, the microarray can be scanned to determine which spots interact with the target. The polynucleotides in the target hybridize with the spots that contain complimentary probe polynucleotides. The presence of hybridized target molecules is then detected by a microarray reader which reports the position and intensity of the label emissions. The identity of the probes which hybridize to the target can be determined by mapping the reported location to records indicating which probe was placed in the reported location.
Microarrays are typically fabricated using one of two techniques: photolighography and robotic spotting. Detailed instructions for using photolithography can be found in U.S. Pat. Nos. 5,445,934 and 5,744,305 which are both incorporated by reference as if set forth fully herein. The photolithographic technique takes approaches used in the design of integrated circuits and applies them in this biological context. While this technique has very high initial costs, it can be used to mass produce arrays at a low incremental cost. Robot spotting techniques are provided in U.S. Pat. No. 5,807,522 which is incorporated by reference as if set forth fully herein. This technique uses a robot to place the probes on the substrate. Techniques used include the use of a pin, capillaries, and inkjet technology. Depending on the technology used, the probes can be manufactured on the substrate or can be manufactured off site and then placed on the substrate. Robotic technology has a lower startup cost but can take time to create a single array. Additionally, the incremental cost per array is higher.
The design of the microarray probe sets has been discussed in detail and commercial software is readily available to perform this task. One such program is explained in Xu, Bioinformatics 2002 Vol. 18, Pages 1432-1437.
Once the data is captured, a number of software programs are available to perform analysis on the resulting data. An example of such software is the TM4 suite of tools available at www.tm4.org.
Selective Breeding Techniques
Selective breeding is the preferential breeding of animals with desired traits in order to increase the prevalence of the trait within the breeding herd. Before starting with selective breeding, a desired goal should be identified. In this case, the trait is SCID, which is not desired in commercial production and thus one goal is to remove the mutations causing SCID in commercial pigs. In order to decrease the frequency of the mutant Artemis alleles, and more particularly the 12 and 16 haplotypes, it is desirable to use selective breeding to decrease the frequency of the allele within a herd. By decreasing the frequency of the allele, fewer animals in the herd will have SCID.
It is also possible that it is of interest to create a herd with a high frequency of the SCID mutation(s) to provide animals that can serve as biomedical models. Thus in some situations, the goal would be to increase the frequency of the SCID mutation in a herd.
Once the goals have been identified, selection and mating are used to produce animals which carry only genes without mutations causing SCID in commercial populations, or which carry one or two copies of the SCID mutation for biomedical purposes.
In both cases, the technology described herein can be used. For elimination of the SCID mutation(s) in a commercial herd, the PCR assays described below would be used to identify animals with a single copy of a mutant gene. Those animals would be removed from the breeding herd; i.e., would NOT be selected for future breeding. Aggressive use of this technique would eliminate the mutant gene in one or two generations. If SCID carrier animals have otherwise useful genetics and are needed for other breeding goals at a commercial breeding company, such carrier animals could be used, but their offspring would again have to be tested for carrying the mutant SCID genes. For creation of a herd for biomedical purposes that would have a high frequency of the mutant genes, marker-assisted introgression (MAI) would be used. MAI is the movement of a target gene or genes from one breed or species (the donor species) into the gene pool of another breed or species (the recipient species) by the repeated backcrossing of a carrier with pigs representing the genetic population into which the SCID mutation is desired. With each repeated round of backcrossing, the amount of non-target genes from the donor line is decreased. As such, depending on the permissible amount of donor genetic material, backcrossing can be conducted for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more generations. Detailed procedures for performing introgression are well known in the art and are discussed in Frisch M, Melchinger A E, Genetics. 2005 June; 170(2):909-17 which is incorporated by reference in its entirety as if set forth fully herein. The offspring of each backcross would be tested for the SCID mutation using the technology described herein, and carrier animals selected for the next backcross mating. Over time, the SCID mutation would be introgressed into the new genetic line.
According to an aspect of the invention, methods for improving production of SCID piglets are provided. In one aspect, pigs with SCID according to the invention may be provided with a bone marrow transfer, thereby reconstituting the immune system of pigs that are homozygous for SCID mutations, such that they can be raised to breeding age, and then mated with SCID carrier pigs or other SCID homozygous pigs. Procedures for performing bone marrow transfers are well known in the art.
The invention also includes novel nucleotide and protein sequences which are causative of SCID. This molecular information can be used in a variety of methods for studying the effects of, the causes of, and possibly the prevention of SCID.
In another embodiment, the invention comprises a method for identifying a genetic marker for SCID in a particular line, strain, breed, population or animal.
It is also possible to establish linkage between specific alleles of alternative DNA markers and alleles of DNA markers known to be associated with a particular gene (e.g. the Artemis gene discussed herein), which have previously been shown to be associated with a particular trait. Thus, in the present situation, taking the Artemis gene, it would be possible, to eliminate animals likely to exhibit SCID or to select for animals likely to exhibit SCID by identifying animals carrying certain alleles of an Artemis associated marker or the Artemis mutations themselves through the use of the allele-specific technology described herein. As used herein, the term “genetic marker” shall include not only the polymorphism disclosed by any means of assaying for the protein changes associated with the polymorphism, but also linked markers, use of microsatellites, or even other means of assaying for the causative protein changes indicated by the marker and the use of the same to influence SCID in an animal.
Use of SCID Pigs
In one aspect, the pigs and cells of the present invention may be used to study a variety of diseases and conditions. Examples of such diseases or conditions include, but are not limited to, endogenous cancers; human immune function, including human autoimmune and immune-mediated diseases; human infectious agents and disease; human vaccine efficacy; human stem cells and regenerative therapeutics; hematopoietic stem cell transplantation and associated diseases; and/or porcine immune function.
The efficacy of the compositions in treating or preventing a particular disease, disorder, or condition using the cells or pigs of the present invention can be evaluated both in vitro and in vivo. As used herein, the term “treating” refers to: (i) preventing a disease, disorder or condition from occurring in a mammal, animal or human that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and/or (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition. For example, with respect to SCID, the ability to respond to infection and/or cancers, or to augment other treatments such as bone marrow transplant or adoptive transfer of immune effectors can be assessed. Further, SCID pigs of the present invention may be used to assess the efficacy of compositions in treating or preventing a particular disease, disorder, or condition using the SCID pigs or cells of the invention as a model organism. The efficacy of such compositions treatment may be measured quantitatively or qualitatively to determine the presence/absence of the disease, or its progression or regression using, in the example of cancer, reduction in tumor size, a reduction in the rate of metastasis, and/or a slowing of tumor growth, and/or no worsening in disease over a specified period of time or other symptoms associated with the disease or clinical indications associated with the pathology of cancer development. In one aspect, this screening may be accomplished using a xenograft transplant, or xenotransplant, porcine SCID model.
All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated as incorporated by reference.
Inhibition, Treatment, and Amelioration of SCID
In one aspect, the invention encompasses methods and compositions for inhibition, treatment, and amelioration of SCID. Compositions include compounds, effectors, and constructs that replace or augment defective Artemis expression or function in SCID affected individuals, or correct the genetic defect causing SCID in affected individuals. In one aspect, the compositions comprise the novel DNA or proteins sequences of the invention. In another aspect, compositions may be pharmaceutical compositions that include Artermis protein or polynucleotides encoding Artermis protein, wherein the Artemis protein has nuclease activity.
In another aspect, the invention encompasses methods for inhibiting, treating, or ameliorating SCID. These methods may include providing or administering Artemis protein or polynucleotides encoding Artemis protein, wherein the Artemis protein has nuclease activity. In one embodiment, the Artemis protein has or is homologous to SEQ ID NO:2, 4, or 6. In another embodiment, the methods utilize polynucleotides encoding proteins having or homologous to SEQ ID NO:2, 4, or 6. In one aspect, the polynucleotides have or are homologous to SEQ ID NO:1, 3, 5, or 7-10.
Pharmaceutical Compositions
According to one aspect of the invention, compositions containing Artemis protein or polynucleotides encoding Artemis protein may be administered to treat SCID. Compositions may be formulated in any conventional manner. Proper formulation is dependent upon the route of administration chosen. Suitable routes of administration include, but are not limited to, oral, parenteral (e.g., intravenous, intraarterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical (nasal, transdermal, intraocular), intravesical, intrathecal, enteral, pulmonary, intralymphatic, intracavital, vaginal, transurethral, intradermal, aural, intramammary, buccal, orthotopic, intratracheal, intralesional, percutaneous, endoscopical, transmucosal, sublingual and intestinal administration.
Pharmaceutically acceptable carriers for use in the compositions of the present invention are well known to those of ordinary skill in the art and are selected based upon a number of factors: concentration and intended bioavailability of the protein or polynucleotide; the disease, disorder or condition being treated with the composition; the subject, his or her age, size and general condition; and the route of administration. Suitable carriers are readily determined by one of ordinary skill in the art (see, for example, J. G. Nairn, in: Remington's Pharmaceutical Science (A. Gennaro, ed.), Mack Publishing Co., Easton, Pa., (1985), pp. 1492-1517, the contents of which are incorporated herein by reference).
The Artemis protein or polynucleotide containing compositions of the present invention may be preferably formulated for parenteral administration, e.g., formulated for injection via intravenous, intraarterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal routes. The compositions of the invention for parenteral administration comprise an effective amount of hydroxytyrosol in a pharmaceutically acceptable carrier. Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions or any other dosage form which can be administered parenterally. Techniques and compositions for making parenteral dosage forms are known in the art. For oral administration, the hydroxytyrosol containing compositions are preferably formulated as tablets, dispersible powders, pills, capsules, gelcaps, caplets, gels, liposomes, granules, solutions, suspensions, emulsions, syrups, elixirs, troches, dragees, lozenges, or any other dosage form which can be administered orally. Techniques and compositions for making oral dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976).
Suitable carriers used in formulating liquid dosage forms for oral or parenteral administration include nonaqueous, pharmaceutically-acceptable polar solvents such as oils, alcohols, amides, esters, ethers, ketones, hydrocarbons and mixtures thereof, as well as water, saline solutions, dextrose solutions (e.g., DW5), electrolyte solutions, or any other aqueous, pharmaceutically acceptable liquid. Suitable nonaqueous, pharmaceutically-acceptable polar solvents include, but are not limited to, alcohols (e.g., .alpha.-glycerol formal, .beta.-glycerol formal, 1,3-butyleneglycol, aliphatic or aromatic alcohols having 2-30 carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, t-butanol, hexanol, octanol, amylene hydrate, benzyl alcohol, glycerin (glycerol), glycol, hexylene glycol, tetrahydrofurfuryl alcohol, lauryl alcohol, cetyl alcohol, or stearyl alcohol, fatty acid esters of fatty alcohols such as polyalkylene glycols (e.g., polypropylene glycol, polyethylene glycol), sorbitan, sucrose and cholesterol); amides (e.g., dimethylacetamide (DMA), benzyl benzoate DMA, dimethylformamide, N-(.beta.-hydroxyethyl)-lactamide, N,N-dimethylacetamide amides, 2-pyrrolidinone, 1-methyl-2-pyrrolidinone, or polyvinylpyrrolidone); esters (e.g., 1-methyl-2-pyrrolidinone, 2-pyrrolidinone, acetate esters such as monoacetin, diacetin, and triacetin, aliphatic or aromatic esters such as ethyl caprylate or octanoate, alkyl oleate, benzyl benzoate, benzyl acetate, dimethylsulfoxide (DMSO), esters of glycerin such as mono, di, or tri-glyceryl citrates or tartrates, ethyl benzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan, fatty acid derived PEG esters, glyceryl monostearate, glyceride esters such as mono, di, or tri-glycerides, fatty acid esters such as isopropyl myristrate, fatty acid derived PEG esters such as PEG-hydroxyoleate and PEG-hydroxystearate, N-methylpyrrolidinone, pluronic 60, polyoxyethylene sorbitol oleic polyesters such as poly(ethoxylated)30-60 sorbitol poly(oleate)2-4, poly(oxyethylene)15-20 monooleate, poly(oxyethylene)15-20 mono 12-hydroxystearate, and poly(oxyethylene)15-20 mono ricinoleate, polyoxyethylene sorbitan esters such as polyoxyethylene-sorbitan monooleate, polyoxyethylene-sorbitan monopalmitate, polyoxyethylene-sorbitan monolaurate, polyoxyethylene-sorbitan monostearate, and Polysorbate® 20, 40, 60 or 80 from ICI Americas, Wilmington, Del., polyvinylpyrrolidone, alkyleneoxy modified fatty acid esters such as polyoxyl 40 hydrogenated castor oil and polyoxyethylated castor oils (e.g., Cremophor® EL solution or Cremophor® RH 40 solution), saccharide fatty acid esters (i.e., the condensation product of a monosaccharide (e.g., pentoses such as ribose, ribulose, arabinose, xylose, lyxose and xylulose, hexoses such as glucose, fructose, galactose, mannose and sorbose, trioses, tetroses, heptoses, and octoses), disaccharide (e.g., sucrose, maltose, lactose and trehalose) or oligosaccharide or mixture thereof with a C4-C22 fatty acid(s)(e.g., saturated fatty acids such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid and stearic acid, and unsaturated fatty acids such as palmitoleic acid, oleic acid, elaidic acid, erucic acid and linoleic acid)), or steroidal esters); alkyl, aryl, or cyclic ethers having 2-30 carbon atoms (e.g., diethyl ether, tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethyl ether); glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether); ketones having 3-30 carbon atoms (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatic hydrocarbons having 4-30 carbon atoms (e.g., benzene, cyclohexane, dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane, sulfolane, tetramethylenesulfon, tetramethylenesulfoxide, toluene, dimethylsulfoxide (DMSO), or tetramethylenesulfoxide); oils of mineral, vegetable, animal, essential or synthetic origin (e.g., mineral oils such as aliphatic or wax-based hydrocarbons, aromatic hydrocarbons, mixed aliphatic and aromatic based hydrocarbons, and refined paraffin oil, vegetable oils such as linseed, tung, safflower, soybean, castor, cottonseed, groundnut, rapeseed, coconut, palm, olive, corn, corn germ, sesame, persic and peanut oil and glycerides such as mono-, di- or triglycerides, animal oils such as fish, marine, sperm, cod-liver, haliver, squalene, squalane, and shark liver oil, oleic oils, and polyoxyethylated castor oil); alkyl or aryl halides having 1-30 carbon atoms and optionally more than one halogen substituent; methylene chloride; monoethanolamine; petroleum benzin; trolamine; omega-3 polyunsaturated fatty acids (e.g., alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid); polyglycol ester of 12-hydroxystearic acid and polyethylene glycol (Solutol® HS-15, from BASF, Ludwigshafen, Germany); polyoxyethylene glycerol; sodium laurate; sodium oleate; or sorbitan monooleate.
EXAMPLES
Embodiments of the present invention are further defined in the following non-limiting Examples. It should be understood that these Examples, while indicating certain embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
Example 1: Initial Characterization of SCID Pigs
The SCID defect was identified in a line of pigs that was initially (1997) derived from pigs that were sourced from private pig breeders in the Midwest, primarily Iowa, and that has since then been selected for increased feed efficiency at Iowa State University. The affected pigs were part of a group of 100 purebred Yorkshire pigs produced after selecting over 7 generations for increased feed efficiency. Parents were partially related, resulting in the offspring having 12-14% inbreeding. The defect was identified in four pigs from this line that died early after challenge in an experimental infection challenge with PRRS virus. The routine necropsy that was conducted on the pigs identified the anomaly, which was confirmed to at least be similar to SCID. Four pigs that were challenged with PRRS virus did not develop an antibody response and, upon necropsy, had both atrophied lymph nodes and thymus. Severe lymphoid hypoplasia/depletion was identified in four 5 to 7 week-old pigs that died unexpectedly or were euthanized because of illness during a porcine reproductive and respiratory syndrome virus (PRRSV) challenge study. Two of the pigs had been inoculated at 4 weeks of age with PRRSV. At necropsy, all four were thin, had rough hair coats, and were smaller than their pen mates. One of the PRRSV inoculated pigs displayed antemortem clinical signs of respiratory disease and had severe fibrinosuppurative bronchopneumonia from which Streptococcus suis was isolated. The thymus was not found in any of the four. Microscopically, there was diffuse, severe lymphoid hypoplasia/depletion with a complete lack of follicles in lymph nodes, tonsils, and Peyer's patches, and an absence of perivascular lymphoid sheaths in the spleen. Immunohistochemical staining for porcine circovirus type-2 was negative for all animals. Immunohistochemical staining revealed scattered T lymphocytes and an absence of B lymphocytes in lymph nodes, spleen, tonsil, and Peyer's patches. Each affected pig was from a different litter, but three had the same sire. Lymphoid tissues from the other 96 pigs in the group, including littermates, were normal. The lesions resemble descriptions of primary immunodeficiency in other species, which have autosomal recessive penetration. This suggests a possible genetic basis to the lymphoid hypoplasia/depletion in these pigs, possibly caused by a recessive mutation.
Unusually low lymphocyte percentages in complete blood counts (CBC) were used to identify additional affected pigs. Using CBC data, >15 affected animals have been identified (all closely related), which upon phenotypic evaluation of the thymus and lymph nodes, as well as immunohistochemistry staining for B cell and T cell markers, have been confirmed as SCID. No effect on neutrophil number was observed. SCID piglets thrive while protected by maternal antibodies from milk, and delayed weaning up to 35 days protects piglets, but piglets at the farm succumb to infections soon thereafter; antibiotics and high biosecurity may extend survival.
Example 2: Phenotypic Characterization of the SCID Phenotype
Flow cytometry (FIG. 1) identified near absence of T and B cells but presence of natural killer (NK) cells in the SCID pigs. The presence and absence of these respective immune cell types was confirmed by quantitative PCR gene expression (FIG. 2). Furthermore, unpublished work at ISU by Dr. J. Cunnick and S. Knetter, in collaboration with Dr. Tuggle, found no clear difference in NK cell activity between SCID and normal pigs but also showed that NK cells could perform their normal killing function only if the cells are stimulated with either IL2, which is expected to be absent in the SCID pigs or IL12/IL18, which may be present in pigs in an inflammatory state. Thus, although our SCID pigs have NK cells, these cells are apparently not functional in vivo.
Finally, assays to quantify the amount of antibody present in umbilical cord blood collected immediately after birth showed much lower levels of total immunoglobulin (Ig) in SCID piglets versus normal littermates (FIG. 3), demonstrating that SCID piglets have a substantially impaired or lack the ability to produce antibodies. Jointly, these findings clearly demonstrate that the affected piglets have SCID.
Example 3: Genetic Basis of SCID
The four piglets that led to the initial discovery of SCID were progeny from four dams and two sires. The four matings that resulted in these piglets were repeated and each mating resulted in at least one SCID piglet, as illustrated in FIG. 4, confirming a genetic basis for the SCID phenotype. The SCID phenotype was found in 35 out of 176 piglets that could be evaluated, i.e. 20%. These 35 pigs were of both sexes as well. The 13 piglets for which the SCID phenotype is unknown died before SCID status was determined. Assuming that they died of SCID, the percentage of SCID progeny would be 48/189=25.4%, suggesting a single recessive autosomal mutation, with parents that produce SCID piglets being carriers.
To map the genomic location of the SCID mutation, a total of 172 pigs were genotyped using the IIlumina 60k Porcine Beadchip. This included 20 affected piglets, their 6 parents, 50 unaffected littermates, and 96 ancestors from the previous 7 generations. Genome-wide Association Analysis of the resulting data using the dfam option of the PLINK software (Purcell et al, 2007), identified a 5.6 Mb region on chromosome 10 that was associated with the SCID phenotype (FIG. 5), with a p-value of 2.7×10−7. Using the Ensemble annotation of the genome, this region was found to contain the Artemis gene, which encodes a ubiquitously expressed protein involved in double-strand DNA break repair processes. Mutations in the Artemis gene in humans have been shown to cause SCID (Moshous et al, 2001).
The SNP genotypes in the 1 Mb region in and around the Artemis gene were then phased into haplotypes, using the program Phase. This included a total of 21 SNPs and resulted in the identification of two separate haplotypes that were associated with the SCID phenotype: the 12 haplotype and the 16 haplotype (FIG. 6). Five of the 6 parents of the initial 4 SCID piglets (FIG. 4) were identified to be carriers of haplotype 16, one parent carried haplotype 12, and the resulting SCID progeny were either homozygous for haplotype 16 or heterozygous 12/16. None of the unaffected progeny were homozygous for 16, homozygous for 12, or heterozygous for 12/16. Subsequently, genetic tests were developed to identify carriers of 12 and 16 haplotypes.
The defect was identified in a line of pigs that was initially (1997) derived from pigs that were sourced from private pig breeders in the Midwest, primarily Iowa, and that has since then been selected for increased feed efficiency. It is unlikely that the described discovery was the result of the selection that was conducted in these lines. More likely, it was either a mutation that occurred in this line or that already existed in the original pigs that created the line and that manifested itself because of some level of inbreeding that occurred in the line. In fact, using phased 60k SNP genotypes of ancestors from the population, the 12 and 16 haplotypes could be traced back from the identified SCIDs to the founder generation of the population, as illustrated in FIG. 7.
Example 4: Molecular Genetic Tests and Basis of the Artemis Mutations Haplotype Tests
In order to identify affected, carrier, and homozygous normal pigs in the ISU population that do not have genotype data, allele-specific primer sets were designed for seven SNPs in the region surrounding the Artemis gene, using Web-based Allele-Specific PCR (WASP; Wangkumhang et al, 2007). PCR cycling conditions for each test included denaturation at 94° C. for 30 seconds (30 s), annealing at temperatures indicated for each primer pair in table 1 for 30 seconds, and elongation at 72° C. for the time indicated in table 1 (E-Time). Each PCR was begun with an initial hot start of 95° C. for 2 minutes (2 m), 40 cycles of amplification, and ended with a final elongation step at 72° C. for 5 m. Table 1 gives the primer sequences, PCR condition information, location, and genotypes for the seven SNPs for which allele specific primers were designed. The bottom portion of the table indicates genotypes, locations of the SNPs, and the number of the SNP in the 1 Mb haplotype from FIG. 6 in parentheses. Three of these SNPs are part of the 21 SNPs in the 1 Mb used for haplotype analysis, as indicated in Table 1. The remaining allele-specific primer sets were designed for SNPs in the 1 Mb upstream of Artemis. These SNPs were chosen as they segregated with the phenotype and the primer design software was able to produce primers sets that were very specific for each allele.







TABLE 1







Primer and PCR conditions, genotypes, location and number of the SNPs.













SNP

Product
FWD Primer
REV Primer
Anneal
E-


Name
Allele
Size
Sequence
Sequence
Temp
Time





ASGA-
A
177
TCCTCTGACCAAGCCTCTG
TCGTCCATGTACCAGAGCC
56
30s


0048074


T (SEQ ID NO: 11)
T (SEQ ID NO: 12)




 



C
177
TCCTCTGACCAAGCCTCTG
CGTCCATGTACCAGAGCC
56
30s





T (SEQ ID NO: 13)
G (SEQ ID NO: 14)




 


ASGA-
A
659
AACCAGTCCCTGACCAACT
TCCATATTTGTTAAGGGCA
54
30s


0048114


G (SEQ ID NO: 15)
GTAATCT 








(SEQ ID NO: 16)




 



G
131
TGCTCAGAGCTTTACATGG
GGCCCATGTTGACATAAA
54
30s





ATTTAG 
GC 







(SEQ ID NO: 17)
(SEQ ID NO: 18)




 


ALGA-
A
643
TCCTCTGCAGGGTTTCAAA
CAGGGTGTGGGACTTTGTT
54
30s


0059043


G (SEQ ID NO: 19)
(SEQ ID NO: 20)




 



G
127
TCAGCTTGGGCAGCTAGG
CCACAGGCACATTGATCTT
54
30s





(SEQ ID NO: 21)
G (SEQ ID NO: 22)




 


H3GA-
A
576
AGTTGAAATCAAAGTATCC
AACTGTAACAAGCGTCCCT
55
30s


0030245


CAA 
TTCT 







(SEQ ID NO: 23)
(SEQ ID NO: 24)




 



C
576
AGTTGAAATCAAAGTATCC
AACTGTAACAAGCGTCCCT
55
30s





CAA 
TTCG 







(SEQ ID NO: 25)
(SEQ ID NO: 26)




 


ALGA-
A
593
GGTATTCTCCTCCTCTACC
CTGGATTGGCAGAGGCTCT
55
30s


059061


TCT 
TTAT 







(SEQ ID NO: 27)
(SEQ ID NO: 28)




 



G
593
GGTATTCTCCTCCTCTACC
CTGGATTGGCAGAGGCTCT
55
30s





TCT 
TTAC 







(SEQ ID NO: 29)
(SEQ ID NO: 30)




 


ALGA-
A
475
GAATGGGAGGTGAGTAAG
CCAGCTGCAAGGGAGACT
55
30s


0059066


TAAA 
(SEQ ID NO: 32)







(SEQ ID NO: 31)





 



C
475
GAATGGGAGGTGAGTAAG
CCAGCTGCAAGGGAGACG
55
30s





TAAA 
(SEQ ID NO: 34)







(SEQ ID NO: 33)





 


ALGA-
A
425
AGCATTAAGACTGTGTGTG
GGTCAAAGTCGTGGGTGT
55
30s


0059080


TGT 
GTTT 







(SEQ ID NO: 35)
(SEQ ID NO: 36)




 



G
425
AGCATTAAGACTGTGTGTG
GGTCAAAGTCGTGGGTGT
55
30s





TGT 
GTTC 







(SEQ ID NO: 37)
(SEQ ID NO: 38)


















ASGA-
ASGA-
ALGA-
H3GA-
ALGA-
ALGA-
ALGA-



0048074
0048114
0059043
0030245
059061
0059066
0059080





Location
51153137
51812252
51975024
52066694
52086867
52109172
52174549



(5)
(16)
(21)






 


h12
C
G
G
C
A
C
A


 


h16
C
A
G
C
A
C
A


 


Normal
A
G
A
A
G
A
G









DNA and cDNA Sequencing to Identify the Causative Mutations

Total RNA was extracted from two sources: whole tissue from ears of 1-day-old piglets, and fibroblasts that were cultured from ear snip tissue. mRNA was reverse transcribed to cDNA. Artemis transcripts were then amplified from the cDNA using the following primers: forward 5′-GGATCCGTGTTCGCCAACGCT-3′ (SEQ ID NO:39) and reverse 5′-GCGGCCGCAGAGCTGCCTTTTAGGTTAT-3′ (SEQ ID NO:40), using an annealing temperature of 60° C. and an extension time of 2 m 30 s. The full Artemis cDNA was amplified using PCR technique, cloned into a TOPO vector, then plasmids transferred into E. coli bacteria which were then grown on LB plates with Ampicillin. Individual colonies were then picked into LB-Ampicillin media and PCR amplified to ensure that each colony contained a vector with Artemis cDNA. Positive PCR products were cleaned using Exo-Sap and sent to the Iowa State University DNA Facility for sequencing. FIG. 8 shows the different transcripts observed in cells from affected pigs. While many types of transcripts were observed, specific parts of the gene are missing for transcripts from the 12 and the 16 haplotypes.
For haplotype 16, all transcripts were found to lack exon 8, which results in a transcript that is 141 bases shorter than the expected full transcript. The sequence and translation of the longest cDNA clone from haplotype 16 is shown in Sequence and Translation 1 (SEQ ID NOS:1 and 2, respectively). Exon 8 and the surrounding region were amplified from genomic DNA extracted from ear snip tissue to investigate the presence or absence of signal sequences required for normal splicing. Exons 7 and 8 and portions of the surrounding introns were amplified using the following primers: forward 5′-CTCAGTGGGTTAGGGACCTG-3′ (SEQ ID NO:41) and reverse 5′-GCCATCTGATAGGGTTTCCA-3′ (SEQ ID NO:42), with annealing temperature of 54° C. and elongation time of 1 m. Animals homozygous for the 16 haplotype were found to have a point mutation of GA at the 615th base of the PCR product (Sequence 4; SEQ ID NO:7). This base is part of the signal sequence for a splice donor site for exon 8, and is required for proper splicing. The A mutation was seen only in homozygous 16 haplotype animals (Sequence 4; SEQ ID NO:7), but a G at this position was seen in the 12 haplotype and all normal haplotypes (Sequence 5; SEQ ID NO:8) that were sequenced.
Transcripts from the 12 haplotype were found to lack a 137 base pair long exon 10, which would cause a frameshift mutation, resulting in a stop codon shortly after the missing exon. Sequence and translation of the longest cDNA clone from haplotype 12 are shown in Sequence and Translation 2 (SEQ ID NOS:3 and 4). Exons 10 and 11 and portions of the surrounding introns were amplified from genomic DNA of homozygous haplotype 12 animals using the following primers: forward 5′-GCTAAAGTCCAGGCCAGTTG-3′ (SEQ ID NO:43) and reverse 5′-CAAGAGTCCCCACCAGTCTT-3′ (SEQ ID NO:44), with annealing temperature of 56° C. and elongation time of 1 m. The signal sequence required for splicing of exon 10 was the same as that seen for normal and reference sequences. However, sequencing of the genomic region around exon 10 identified a nonsense point mutation in the 12 haplotype exon 10 sequence at base 116 of the PCR product (Sequence 6; SEQ ID NO:9), with a mutation from G→A. Under normal transcription, splicing and translation, this mutation from G→A would change a Tryptophan amino acid codon to a stop codon, resulting in a protein that ends with 266 of the total 712 amino acids of the normal Artemis protein. We have not observed such a transcript in 12/12 animal cells thus far. For the 12 haplotype transcript we do observe, the predicted protein translation would be 277 amino acids long (Translation 2; SEQ ID NO:2). The genomic sequences for exons 10 and 11 are shown in sequence 7 (SEQ ID NO:10).
These results for the 12 and 16 haplotype sequencing did not show any transcripts containing all exons expressed in pig cells that were homozygous for 12 or the 16 through rt-PCR amplification using primers at the beginning and end of the normal transcript. Thus a normal Artemis protein cannot be produced from any of the RNAs observed. For the 16 haplotype, a normal amount of RNA should be present in these cells, which will make a protein that missing 47 amino acids. For the 12 haplotype, the protein produced would be severely truncated if a transcript containing exon 10 was produced; however, no evidence that such a transcript is stably produced in 12/12 homozygous animals was found. Translation of the transcript observed would also result in a truncated protein. While >150 different mutated alleles of Artemis have been found through 2010 (Pannicke et al. 2010), none of the reported mutant alleles are exactly the same as the two mutants we have identified or as their predicted RNAs/proteins. Thus, it is difficult to be sure of the actual resulting residual level of activity expressed by these mutations. We anticipate that the protein predicted to be expressed from the 16 mutation may have some residual activity, while the protein predicted to be expressed from the 12 mutation would be completely non-functional.
Genotyping Methods Used for Crossbred Animals
Known carrier pigs were mated to Duroc, Landrace, or crossbred animals in order to introduce a more diverse genetic background into the SCID carrier population. Offspring that resulted from these matings were tested for carrier status (50% of each litter produced expected to be SCID carriers) using the seven sets of allele-specific primers described above. The genetic tests performed were not able to accurately distinguish carriers from homozygous normal piglets. To genotype these crossbred animals, genomic regions containing exons with causative mutations (exon 8 for suspected haplotype 16 carriers and exon 10 for suspected haplotype 12 carriers) were amplified, cleaned using Exo-Sap, then sequenced, as described in section 4b. This method of genotyping is most accurate in animals outside of the ISU Yorkshire population, such as animals of other breeds or commercial animals.
Example 5: Cellular Evidence of Artemis as the Causative Gene
A specific property of mutations in the Artemis gene, compared to mutations in RAG1/2 genes, is that a non-functional Artemis gene causes increased sensitivity of cells to irradiation because Artemis is involved in DNA repair. This was evaluated by irradiation of fibroblasts from SCID piglets and their normal litter mates. Results (FIG. 9) clearly established the increased sensitivity of fibroblasts from the SCID piglets to irradiation, eliminating RAG1/2 genes as causes for the SCID mutation in the ISU pigs of the present invention.
Further, we have demonstrated that the radiosensitivity is due to the lack of Artemis protein activity in the SCID pig-derived fibroblasts. We inserted a plasmid containing the human Artemis cDNA (pArt, which expresses a functional Artemis protein) into fibroblasts from SCID pigs (n=2) or normal animals (n=3). Transfected cells were incubated overnight in cell culture media. One day after transfection, fibroblasts were subjected to 4 Gy irradiation and plated in triplicate. Addition of pArt increased the radioresistance of SCID fibroblasts (p=0.0012), but normal cells were unaffected (p=0.11; FIG. 15). This demonstrates that mutations creating the lack of Artemis function cause the radiosensitivity seen in fibroblasts from SCID pigs, as the addition of Artemis protein can rescue the phenotype.
Example 6: Bone Marrow Transfer Rescues
In order to both establish the SCID as a model for bone marrow transplantation research, as well as rescue SCID affected piglets by providing them with an immune system such that they could be raised to breeding age (FIG. 10), nine affected piglets from four litters were given bone marrow transfers. Marrow donors were matched based on low-density MHC genotyping. All nine piglets were successfully transplanted, however, five of the nine showed signs of graft versus host disease after transfer and were euthanized.
Engraftment and establishment of a functional adaptive immune system was documented in the four successful BMT recipients based on lymphocyte counts (FIG. 10), donor origin of those lymphocytes (FIG. 11), presence of normal major lymphocyte populations based on flow cytometry, and response to vaccination (FIG. 12).
Example 7: Spontaneous Cancer Development in Bone Marrow Transfer SCID Recipients
Three of the four successful BMT SCID recipients developed spontaneous cancers when they were about 1 year of age. (Table 2). The development of these cancers likely is related to the fact that they have a mutation in Artemis, which is involved in DNA repair, and that the BMT SCID pig's genetics were either 16/16 or 12/16 and thus their immune cells may have had some residual activity, leading to aberrant DNA recombination and oncogene activation, which is seen in some human Artemis patients (Moshous et al., 2003 PMID: 12569164) and in some mouse models (Jacobs et al. 2011 PMID: 21147755).







TABLE 2







List of successful BMT transfers and their fate.














Necropsy



Animal ID
Genotype
Necropsy Date
Age
Type of cancer





Cornelius
16/16
Feb. 19, 2013
12 mo 21 d
T cell lymphoma


Maria
16/16
Dec. 31, 2012
11 mo 2 d
Lymphoma/






leukemia


Abby
12/16
May 22, 2013
11 mo 20 d
Nephroblastoma









Wayne
12/16
N/A - Still alive









Example 8: Direct Genetic Tests for the SCID Mutations
FIG. 14 shows the orientation to the two different mutations, their relationship to each other along the chromosome, and the new genetics tests for SCID in pigs.
Specific tests were developed for the mutant sequence at each of the two variants in the Artemis/DCLRE1C gene. For the causative mutation in haplotype 16, found at position 615 of Sequence 4 (SEQ ID NO:7), the test was established for all animals in which it was assessed. For the causative mutation in haplotype 12, found at position 116 of Sequence 6 (SEQ ID NO:9), the test was able to genotype most animals but not all (see information below).
The ABI PRISM® SNaPshotTM Multiplex Kit (Lifetechnologies, P/N 4323151) technology was used for the tests. SNaPshot can be used to distinguish four nucleotides at a specific position in a sequence by color. First, a PCR is performed to amplify the region surrounding the SNP of interest, i.e. one PCR for the region surrounding haplotype 12 (h12) SNP and one for the region surrounding haplotype 16 (h16) SNP. The primers used are called the outside primers for the h12 SNP and for the h16 SNP in Table 3. The melting temperature for both PCRs is Tm=54° C. Next, the PCR products are cleaned from remaining primers and dNTPs through adding 5 units of Shrimp Alkaline Phosphatase (SAP, USB Corporation, P/N 70092) and 2 units of Exonuclease I (ExoI, New England BioLabs, P/N M0293) to every 15 ul of PCR product. To get to the right concentration, ExoI is diluted in a buffer containing 80 mM Tris-HCl and 2 mM MgCl2. The mixture of PCR product, SAP and ExoI is incubated at 37° C. for 1 hour and deactivated at 75° C. for 15 minutes. The cleaned PCR products (one for each SNP) of one animal can thereafter be pooled and analyzed together with the SNaPshotTM Multiplex Kit. This kit encloses a SNaPshot Multiplex Ready Reaction Mix that contains fluorescently labeled ddNTPs and AmpliTaq® DNA polymerase in a reaction buffer. In a reaction volume of 10 ul, we have 5 ul of the SNaPshot Multiplex Ready Reaction Mix, 3 ul of pooled PCR products and 0.2 uM of each extension primer (for sequence see table 1). The extension reaction goes as followed: 25 cycles of 10 minutes at 96° C., 5 minutes at 50° C. and 30 minutes at 60° C. 1 unit of SAP is added after this reaction to remove unincorporated fluorescent ddNTPs from the reaction. This SAP incubation is done at 37° C. for 1 hour and deactivated at 75° C. for 15 minutes. To read the fluorescent signal, this product needs to run on an ABI Prism 3700 DNA Analyzer (or 310 or 3100 Genetic Analyzer), using the GeneScan 120 LIZ size standard, with dye set E5.







TABLE 3







PCR Primers and Oligonucleotides used in SNaPshot Direct


Genetic Tests for the Causative Mutations













Primer
Primer
Primer Sequence
SCID
Normal


Haplotype
Use
Direction
5′-3′
allele
allele





h16
PCR
Forward
CTCAGTGGGTTAGGGACCTG 







(SEQ ID NO: 46)




 




Reverse
GCCATCTGATAGGGTTTCCA 







(SEQ ID NO: 47)




 



SNaPshot
Forward
GAGGAGTTCGGAGTCCAG 
A
G





(SEQ ID NO: 48)




 




Reverse
TCTCTGGGAGAAAGAGCCCTCAGGTA
T
C





(SEQ ID NO: 49)




 


h12
PCR
Forward
GCATTCACTCAGGCTGCTTT 







(SEQ ID NO: 50)




 




Reverse
CCCAGGAAATACTGGCTCAT 







(SEQ ID NO: 51)




 



SNaPshot
Forward
CCCCATCTTTTTTAGGCAGAGGAATATTTTCATT
A
G





(SEQ ID NO: 52)




 




Reverse
CTCTCTCTCTCTCCGGATGTTATTCCACAGGGTAAT
T
C





TTATTC (SEQ ID NO: 53)









Both tests are performed in matings in which the parents contain both the h16 and h12 alleles, in order to correctly determine the combined genotype at both positions. In matings where only one SCID haplotype is segregating, only one of the two tests needs to be performed. We have not observed an animal that carries both mutations on the same chromosome, the two mutations appear to always be on separate chromosomes.

H16 Test (Position 615 of Sequence 4; SEQ ID NO:7)

The h16 test correctly determines the allele at the h16 position (either G/C or A/T) for all possible genotypes. These include a GG result for normal animals at both the h12 and h16 positions, as well as h12/h12 animals, a GA result for h12/h16 and normal/h16 animals, and a AA result for h16/h16 animals (see FIG. 14A).
H12 Test (Position 116 of Sequence 6; SEQ ID NO:9)
The h12 test detects the h12 mutant sequence at position 116 of SEQ ID NO:9 but will not always detect the normal sequence on the other chromosome in a h12/h16 or a h12/normal animal. Recent results from SNaPshot genotyping of h12 mutation carriers have shown that the primers given above may preferentially amplify the h12 mutated allele when present. In the absence of the h12 mutated allele, SNaPshot results show the expected GG (normal/normal at this position) genotype. In certain cases, SNaPshot genotyping of animals with the known genotype of h12/h16 or h12/normal have shown only the h12 mutant allele (AA).
Combining Technologies to Obtain Exact Genotype at the Artemis Locus
Correct identification of genotypes for animals that have resulted in these SNaPshot genotyping errors have been found through the use of the primers discussed in Table 1 of Example 4 above, in addition to SNaPshot results from h16 allele genotyping. The primers of Table 1 of Example 4 are capable of determining whether an animal is SCID affected, carrier, or normal. This information, combined with the genotype from h16 SNaPshot will show the exact genotype of that animal.
Example 8: Sequences
In each nucleic acid sequence, unless otherwise noted, primer sequences are shown in bold, correct translation frames are underlined, location of missing sequences are indicated in parentheses, and premature stop codon are shown in italics. Splice donor site mutation indicated by brackets. Point mutations indicated by curly brackets.








Sequence 1, Transcript of 20101 clone 10, longest cDNA sequence



from haplotype 16


(SEQ ID NO: 1)



GGATCCGTGTTCGCCAACGCTATGAGTTCCTTCGAGGGCCAGATGAGTTCCT



 


TCGAGGGCCAGATGGCGGAGTACCCAACTATCTCCATAGACCGTTTCGACCGG


 


GAGAATCTGAGGGCTCGCGCTTATTTCCTGTCCCACTGCCACAAGGATCACAT


 


GAAAGGATTAAGAGCCTCTACCTTGAGAAGAAGGTTGGAGTGCAGCTTGAAGG


 


TCTCCTTATACTGTTCACCTGTTACTAGAGAATTGTTATTAACCAACCCGAGGT


 


ACAGATTTTGGGAGAAACGAATTGTGTCAATTGAAGTTGAAACTCCTACCCAG


 


ATATCTTTAATTGATGAAGCATCAGGCGAGAAGGAAGAAATTGTTGTGACTCT


 


CTTACCAGCTGGTCATTGCCCAGGATCAGTTATGTTCTTATTTCAGGGCAACAA


 


TGGAACTGTCTTGTATACAGGAGACTTCCGATTGGCAAAAGGAGAAGCTGCCA


 


GAATGGAGCTTCTGCACTCGGGGGGCAGTGTGAAAGACATCCAGAGTGTGTAC


 


TTAGACACCACTTTCTGCCATCCAAAGTATTACCAAATTCCCAGTCGGG(EXON


 


8)TTCACGTGAATAAACTGGACATGTTTCGAAACATGCCTGACATCCTTCATCAT


 


CTCACAACAGACCGTGGCACTCAGATCCATGCCTGTCGGCATCCAAAGGCAGA


 


GGAATATTTTCATTGGAATAAGTTACCCTGTGGAATAACATCCAAAAATAGAA


 


TTCCACTCCACATAATCAGCATTAAGCCCTCCACTATGTGGTTTGGAGAAAGA


 


ACTAGAAAAACCAATGTTATTGTGAGGACTGGAGAGAGTTCGTACAGAGCCTG


 


CTTTTCTTTTCACTCCTCCTACAGTGAGATTAAAGATTTCTTGAGCTACATTAGC


 


CCTGTGAATGTATATCCAAATGTCATTCCACTGGGCACAACTCTGGAGAAAGT


 


TAAAGAAATCTTAAAGCCTTTATGCCGATCTTCGCAAAATATCGAGCCAAAGT


 


ATAAACCACTTGGAAAATTGAAGAGAGCTAGAATAATCCATCTAGACTCAGAG


 


GAGGAGGAGGAGGACGATGACGATCTCTTTGATGATCCTCTGCCAGTACCTTT


 


AAGGCACAAGGTTCCAAATCAGCAGACTCTTCACTCTGAGGTACTTCCCATGA


 


CTGCTCTACCACAAGACCAGCCTGAAAAACAGACAGAAAGCACAGAATGCTTC


 


AAAGCAGAGAGTATGCCAACATGTCTCTGGGCAAACTTCGTAGATTGTGAAGA


 


ATCCAATAGTGAAAGTGAAGAATTAGAAATCACGGCTCCAGCTCAAGGAGAC


 


ACGAGTCCTGTCCCCCATCACCAGCAGAAGGCTGAAGGGGAAGTACCACAGTG


 


GGAAGTGTTCTTTAAAAGAAATGATGAAATCACAGATGACTGTTTGGAAAACC


 


TTCCGTCCTCCACAGAGGCAGGGGGCTCTCAGTCCCCAAAGCTTTTCAGTGACT


 


CTGATGGGGAATCAACTCACATTTCTTCCCAGACTTCTTCTCAGTCAACACACA


 


TATCAGAACAAGGAAGTCAAGGCTGGGACAGCCAATCAGACACTGTTTTGTTA


 


TCTTCCCAAGAGAGAAAAAGTGGGGATATTACCTCCTTGAACAAAGGTGGCTC


 


TAGACCAGAAATCAAAGAGAATATTCCCATCCTTCAGATGGAACAAAATGTAT


 


TTTGCCCGAAGGATACTTACTCTGATTTGAAAGGCAGAGATCAAGATATAAAC


 


ACACTTCCCAGTGCTAGAGAAACAACTACTCTGAGCAGTGGGAAACACATGCC


 


TCAGGAGAAAAGGCCGCTAAACTGTAACAGTAACACAGATTCACAAGGCTCCT


 


CTGACTTTGAAATTCCCTCCACTCCAGAAGCTGAGCTACCTCAACAAGAGCAT


 


CTGCAATATTTATACAAGAAGTTGGCAGGAGGAGAGGGTATAGTAATTGAAAA


 


AAGGAAAAGCGCACGTCATTCTAGAGCAACCATTAAAAAACCTACACAAACA


 


GGTAATAGTCAGACTCCTAATAGATGAGTTCAAATGGAGTACTTAAAAATGTTC


 


ATATAACCTAAAAGGCAGCTCTGCGGCCGC


 


Translation 1 


(SEQ ID NO: 2)



MSSFEGQMSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRASTLR



 


RRLECSLKVSLYCSPVTRELLLTNPRYRFWEKRIVSIEVETPTQISLIDEASGEKEEIV


 


VTLLPAGHCPGSVMFLFQGNNGTVLYTGDFRLAKGEAARMELLHSGGSVKDIQSV


 


YLDTTFCHPKYYQIPSRVHVNKLDMFRNMPDILHHLTTDRGTQIHACRHPKAEEYF


 


HWNKLPCGITSKNRIPLHIISIKPSTMWFGERTRKTNVIVRTGESSYRACFSFHSSYS


 


EIKDFLSYISPVNVYPNVIPLGTTLEKVKEILKPLCRSSQNIEPKYKPLGKLKRARIIH


 


LDSEEEEEDDDDLFDDPLPVPLRHKVPNQQTLHSEVLPMTALPQDQPEKQTESTEC


 


FKAESMPTCLWANFVDCEESNSESEELEITAPAQGDTSPVPHHQQKAEGEVPQWE


 


VFFKRNDEITDDCLENLPSSTEAGGSQSPKLFSDSDGESTHISSQTSSQSTHISEQGSQ


 


GWDSQSDTVLLSSQERKSGDITSLNKGGSRPEIKENIPILQMEQNVFCPKDTYSDLK


 


GRDQDINTLPSARETTTLSSGKHMPQEKRPLNCNSNTDSQGSSDFEIPSTPEAELPQ


 


QEHLQYLYKKLAGGEGIVIEKRKSARHSRATIKKPTQTGNSQTPNR-


 


Sequence 2, Transcript of 20301 clone 1, longest cDNA sequence


from haplotype 12 


(SEQ ID NO: 3)



GGATCCGTGTTCGCCAACGCTATGAGTTCCTTCGAGGGCCAGATGGCGGAGT



 


ACCCAACTATCTCCATAGACCGTTTCGACCGGGAGAATCTGAGGGCTCGCGCT


 


TATTTCCTGTCCCACTGCCACAAGGATCACATGAAAGGATTAAGAGCCTCTAC


 


CTTGAAAAGAAGGTTGGAGTGCAGCTTGAAGGTCTCCTTATACTGTTCACCTGT


 


TACTAGAGAATTGTTATTAACCAACCCGAGGTACAGATTTTGGGAGAAACGAA


 


TTGTGTCAATTGAAGTTGAAACTCCTACCCAGATATCTTTAATTGATGAAGCAT


 


CAGGCGAGAAGGAAGAAATTGTTGTGACTCTCTTACCAGCTGGTCATTGCCCA


 


GGATCAGTTATGTTCTTATTTCAGGGCAACAATGGAACTGTCTTGTATACAGGA


 


GACTTCCGATTGGCAAGAGGAGAAGCTGCCAGAATGGAGCTTCTGCACTCGGG


 


GGGCAGTGTGAAAGACATCCAGAGTGTGTACTTAGACACCACTTTCTGCCATC


 


CAAAGTATTACCAAATTCCCAGTCGGGAGGAGTGTCTGAGAGGGATCTTGGAG


 


CTGGTCCGCAGCTGGATCACACGGAGCCCCTACCACGTGGTGTGGCTGAACTG


 


CAAAGCGGCCTATGGGTACGAGTACCTGTTCACCAACCTCAGCGAGGAGTTCG


 


GAGTCCAGGTTCACGTGAATAAACTGGACATGTTTCGAAACATGCCTGACATC


 


CTTCATCATCTCACAACAGACCGTGGCACTCAGATCCATGCCTGTCGGCATCCA


 


AAG(EXON10)AACTGGAGAGAGTTCGTACAGAGCCTGCTTTTCTTTTCACTCCT


 


CCTACAGTGAGATTAAAGATTTCTTGAGCTACATTAGTCCTGTGAATGTATATC


 


CAAATGTCATTCCACTGGGCACAACTCCGGAGAAAGTTAAAGAAATCTTAAAG


 


CCTTTATGCCGATCTTCGCAAAATATCGAGCCAAAGTATAAACCACTTGGAAA


 


ATTGAAGAGAGCTAGAATAATCCATCTAGACTCAGAGGAGGAGGAGGAGGAC


 


AATGACGATCTCTTTGATGATCCTCTGCCAGTACCTTTAAGGCACAAGGTTCCA


 


AATCAGCAGACTCTTCACTCTGAGGTACTTCCCATGACTGCTCTACCACAAGAC


 


CAGCCTGAAAAACAGACAGAAAGCACAGAATGCTTCAAAGCAGAGAGTATGC


 


CAACATGTCTCTGGGCAAACTTCGTAGATTGTGAAGAATCCNATAGTGAAAGT


 


GAAGAATTAGAAATCACGGCTCCAGCTCAAGGAGACACGAGTCCTGTCCCCCA


 


TCACCAGCAGAAGGCTGAAGGGGAAGTACCACAGTGGGAAGTGTTCTTTAAAA


 


GAAATGATGAAATCACAGATGACTGTTTGGAAAACCTTCCGTCCTCCACAGAG


 


GCAGGGGGCTCTCAGTCCCCAAAGCTTTTCAGTGACTCTGATGGGGAATCAAC


 


TCACATTTCTTCCCAGACTTCTTCTCAGTCAACACACATATCAGAACAAGGAAG


 


TCAAGGCTGGGACAGCCAATCAGACACTGTTTTGTTATCTTCCCAAGAGAGAA


 


AAAGTGGGGATATTACCTCCTTGAACAAAGGTGGCTCTAGACCAGAAATCAAA


 


GAGAATATTCCCATCCTTCAGATGGAACAAAATGTATTTTGCCCGAAGGATAC


 


TTACTCTGATTTGAAAGGCAGAGATCAAGATATAAACACACTTCCCAGTGCTA


 


GAGAAACAACTACTCTGAGCAGTGGGAAACACATGCCTCAGGAGAAAAGGCC


 


GCTAAACTGTAACAGTAACACAGATTCACAAGGCTCCTCTGACTTTGAAATTC


 


CCTCCACTCCAGAAGCTGAGCTACCTCAACAAGAGCATCTGCAATATTTATAC


 


AAGAAGTTGGCAGGAGGAGAGGGTATAGTAATTGAAAAAAGGAAAAGCGCAC


 


GTCATTCTAGAGCAACCACTAAAAAACCTACACAAACAGGTAATAGTCGGACT


 


CCTAATAGATGAGTTCAAATGGAGTACTTAAAAATGTTCATATAACCTAAAAG


 


GCAGCTCTGCGGCCGC


 


Translation 2 


(SEQ ID NO: 4)



MSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRASTLKRRLECSLK



 


VSLYCSPVTRELLLTNPRYRFWEKRIVSIEVETPTQISLIDEASGEKEEIVVTLLPAGH


 


CPGSVMFLFQGNNGTVLYTGDFRLARGEAARMELLHSGGSVKDIQSVYLDTTFCH


 


PKYYQIPSREECLRGILELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEEFGVQ


 


VHVNKLDMFRNMPDILHHLTTDRGTQIHACRHPKNWREFVQSLLFFSLLLQ-


 


Sequence 3 Transcript of 20106 clone 6, longest cDNA sequence


from normal haplotype


(SEQ ID NO: 5)



GGATCCGTGTTCGCCAACGCTATGAGTTCCTTCGAGGGCCAGATGGCGGAGT



 


ACCCAACTATCTCCATAGACCGTTTCGACCGGGAGAATTTGAGGGCTCGCGCT


 


TATTTCCTGTCCCACTGCCACAAGGATCACATGAAAGGATTAAGAGCCTCTAC


 


CTTGAAAAGAAGGTTGGAGTGCAGCTTGAAGGTCTCCTTATACTGTTCACCTGT


 


TACTAGAGAATTGTTATTAACCAACCCGAGGTACAGATTTTGGGAGAAACGAA


 


TTGTGTCAATTGAAGTTGAAACTCCTACCCAGATATCTTTAATTGATGAAGCAT


 


CAGGCGAGAAGGAAGAAATTGTTGTGACTCTCTTACCAGCTGGTCATTGCCCA


 


GGATCAGTTATGTTCTTATTTCAGGGCAACAATGGAACTGTCTTGTATACAGGA


 


GACTTCCGATTGGCAAAAGGAGAAGCTGCCAGAATGGAGCTTCTGCACTCGGG


 


GGGCAGTGTGAAAGACATCCAGGGTGTGTACTTAGACACCACTTTCTGCCATC


 


CAAAGTATTACCAAATTCCCAGTCGGGAGGAGTGTCTGAGAGGGATCTTGGAG


 


CTGGTCCGCAGCTGGATCACACGGAGCCCCTACCACGTGGTGTGGCTGAACTG


 


CAAAGCGGCCTATGGGTACGAGTACCTGTTCACCAACCTCAGCGAGGAGTTCG


 


GAGTCCAGGTTCACGTGAATAAACTGGACATGTTTCGAAACATGCCTGACATC


 


CTTCATCATCTCACAACAGACCGTGGCACTCAGATCCATGCCTGTCGGCATCCA


 


AAGGCAGAGGAATATTTTCATTGGAATAAATTACCCTGTGGAATAACATCCAA


 


AAATAGAATTCCACTCCACATAATCAGTATTAAGCCCTCCACTATGTGGTTTGG


 


AGAAAGAACTAGAAAAACCAATGTTATTGTGAGAACTGGAGAGAGTTCGTAC


 


AGAGCCTGCTTTTCTTTTCACTCCTCCTACAGTGAGATTAAAGATTTCTTGAGC


 


TACATTAGTCCTGTGAATGTATATCCAAATGTCATTCCACTGGGCACAACTCTG


 


GAGAAAGTTAAAGAAATCTTAAAGCCTTTATGCCGATCTTCGCAAAATATCGA


 


GCCAAAGTATAAACCACTTGGAAAATTGAAGAGAGCTAGAATAATCCATCTAG


 


ACTCAGAGGAGGAGGAGGAGGACAATGACGATCTCTTTGATGATCCTCTGCCA


 


GTACCTTTAAGGCACAAGGTTCCAAATCAGCAGACTCTTCACTCTGAGGTACTT


 


CCCATGACTGCTCTACCACAAGACCAGCCTGAAAAACAGACAGAAAGCACAG


 


AATGCTTCAAAGCAGAGAGTATGCCAACATGTCTCTGGGCAAACTTCGTAGAT


 


TGTGAAGAATCCAATAGTGAAAGTGAAGAATTAGAAATCACAGCTCCAGCTCA


 


AGGAGACACGAGTCCTGTCCCCCATCACCAGCAGAAGGCTGAAGGGGAAGTA


 


CCACAGTGGGAAGTGTTCTTTAAAAGAAATGATGAAATCACAGATGACTGTTT


 


GGAAAACCTTCCGTCCTCCACAGAGGCAGGGGGCTCTCAGTCCCCAAAGCTTT


 


TCAGTGACTCTGATGGGGAATCAACTCACATTTCTTCCCAGACTTCTTCTCAGT


 


CAACACACATATCAGAACAAGGAAGTCAAGGCTGGGACAGCCAATCAGACAC


 


TGTTTTGTTATCTTCCCAAGAGAGAAAAAGTGGGGATATTACCTCCTTGAACAA


 


AGGTGGCTCTAGACCAGAAATCAAAGAGAATATTCCCATCCTTCAGATGGAAC


 


AAAATGTATTTTGCCCGAAGGATACTTACTCTGATTTGAAAGGCAGAGATCAA


 


GATATAAACACACTTCCCAGTGCTAGAGAAACAACTACTCTGAGCAGTGGGAA


 


ACACATGCCTCAGGAGAAAAGGCCGCTAAACTGTAACAGTAACACAGATTCAC


 


AAGGCTCCTCTGACTTTGAAATTCCCTCCACTCCAGAAGCTGAGCTACCTCAAC


 


AAGAGCATCTGCAATATTTATACAAGAAGTTGGCAGGAGGAGAGGGTATAGTA


 


ATTGAAAAAAGGAAAAGCGCACGTCATTCTAGAGCAACCACTAAAAAACCTA


 


CACAAACAGGTAATAGTCAGACTCCTAATAGATGAGTTCAAATGGAGTACTTA


 


AAAATGTTCATATAACCTAAAAGGCAGCTCTGCGGCCGC


 


Translation 3 


(SEQ ID NO: 6)



MSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRASTLKRRLECSLK



 


VSLYCSPVTRELLLTNPRYRFWEKRIVSIEVETPTQISLIDEASGEKEEIVVTLLPAGH


 


CPGSVMFLFQGNNGTVLYTGDFRLAKGEAARMELLHSGGSVKDIQGVYLDTTFCH


 


PKYYQIPSREECLRGILELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEEFGVQ


 


VHVNKLDMFRNMPDILHHLTTDRGTQIHACRHPKAEEYFHWNKLPCGITSKNRIPL


 


HIISIKPSTMWFGERTRKTNVIVRTGESSYRACFSFHSSYSEIKDFLSYISPVNVYPNV


 


IPLGTTLEKVKEILKPLCRSSQNIEPKYKPLGKLKRARIIHLDSEEEEEDNDDLFDDPL


 


PVPLRHKVPNQQTLHSEVLPMTALPQDQPEKQTESTECFKAESMPTCLWANFVDC


 


EESNSESEELEITAPAQGDTSPVPHHQQKAEGEVPQWEVFFKRNDEITDDCLENLPS


 


STEAGGSQSPKLFSDSDGESTHISSQTSSQSTHISEQGSQGWDSQSDTVLLSSQERKS


 


GDITSLNKGGSRPEIKENIPILQMEQNVFCPKDTYSDLKGRDQDINTLPSARETTTLS


 


SGKHMPQEKRPLNCNSNTDSQGSSDFEIPSTPEAELPQQEHLQYLYKKLAGGEGIVI


 


EKRKSARHSRATTKKPTQTGNSQTPNR-


 


Sequence 4, Genomic sequence of exons 7 and 8 (dotted  


underlined) of 4810, a homozygous haplotype 16 


affected animal 


(SEQ ID NO: 7)



TCCCTCACCCATCTGTCTTATATATTATCCTATGGAAATCACTTTCTAACCATGT



 


CGATACAGTAAAAAATTCCAGGGAGCTCTCTTGTGGCCCAGTGGATTAAGGAT


 


CTGGCTGTTGTCACTGCAGCGGCGTGGGAGTGGCCAAAAAAGAAAACCCAAGT


 


GGACTGAAACCTCCTGTAAAATAACTGAGAGTTTCGACTTCACGCGTGTTGTAT


 








Sequence 5, genomic sequence of exons 7 and 8 (dotted  


underline) of 1304, a homozygous normal animal 


(SEQ ID NO: 8)



TCCCTCACCCATCTGTCTTATATATTATCCTATGGAAATCACTTCCTAACCATGTCG



 


ATACAGTAAAAAATTCCAGGGAGCTCTCTTGTGGCCCAGTGGATTAAGGATCTGG


 


CTGTTGTCACTGCAGCGGCATGGGAGTGGCCAAAAAAGAAAACCCAAGTGGACTG


 


AAACCTCCTGTAAAATAACTGAGAGTTTCGACTTCACGCGTGTTGTATTTCACGTG


 








Sequence 6, Genomic sequence of exons 10 and 11 


(underlined)  of 20301, a homozygous haplotype 12 animal


(SEQ ID NO: 9)



CATGCTAAAAAGTCATCTGCATTTTTTTGAGACCNTGGTACAAATATATTTGTT



 








CTTTGAGGTAAATCGATAGTTTAAAGGCAGTCTAGCCTAACCTCAAGAGGGGG


 


GCATATCATGATTGTGGAAAATAACTTTTGAAAGTTAAACTCTGTTTAAATTAA


 


AGAGAAGCGGCTTCCAAAAGCTATCAATTGCTTACTACCATGAGCCAGTATTT


 


CCTGGGGTCTACTGAGTTTGACTGAGAAGATGGTTAGAGGCAGGCTGTCTTGC


 


TTGGCCGGAAGTTGGGAATTGACCTTGAAGGGGTTGGAAGCCCTTAGTGGAGC


 


AGAGGAGGCCCATACTGATCCTCATGTTTTAGCTCATGTCCTGAAACATTGGCC


 


GTTTGGAATGTTCACAGATATTTACATTTATGGAAAGAGTTCCCTCCTGGCCCA


 


GGAAAAAAATCCTTGATGGCAGGGTGATTTCATCCTGCAGTAGCCTAAAATGA


 


GAAGACTCGGAGTTTGGAACTTAGTTTTGATGAGTGACCCTTAATTTTGGGTTT


 








Sequence 7, Genomic sequence of exons 10 and 11 


(underlined) of 1304, a homozygous normal animal 


(SEQ ID NO: 10)



CATGCTAAAAAGTCATCTGCATTTTTTTGAGACCCTGGTACAAATATATTTGTTGA



 








TCGATAGTTTAAAGGCAGTCTAGCCTAACCTCAAGAGGGGGGCATATCATGATTG


 


TGGAAAATAACTTTTGAAAGTTAAACTCTGTTTAAATTAAAGAGAAGCGGCTTCCA


 


AAAGCTATCAATTGCTTACTACCATGAGCCAGTATTTCCTGGGGTCTACTGAGTTT


 


GACTGAGAAGATGGTTAGAGGCAGGCTGTCTTGCTTGGCCGGAAGTTGGGAATTG


 


ACCTTGAAGGGGTTGGAAGCCCTTAGTGGAGCAGAGGAGGCCCATACTGATCCTC


 


ATGTTTTAGCTCATGTCCTGAAACATTGGCCGTTTGGAATGTTCACAGATATTTAC


 


ATTTATGGAAAGAGTTCCCTCCTGGCCCAGGAAAAAAATCCTTGATGGCAGGGTG


 


ATTTCATCCTGCAGTAGCCTAAAATGAGAAGACTCGGAGTTTGGAACTTAGTTTTG


 








Porcine DCLRE1C (Artemis) gene sequence (SEQ ID NO: 45): Exon 


regions are underlined; the haplotype 12 and 16 causative


mutations are boxed in light grey and dark grey, respectively.


>ENSSSCG00000011049|ENSSSCT00000012093


ATAACCGCGGAGGGCTGGCGCCCAGTCGGCTGTGTTCGCCAACGCTATGAGTT


 


CCTTCGAGGGCCAGATGGCGGAGTACCCAACTATCTCCATAGACCGTTTCGAC


 


CGGGAGAATCTGAGGGCTCGCGCTTATTTCCTGTCCCACTGCCACAAGGGTGA


 


GTGAGCGCGGCGCGCCGACCCCCTCCCGGGGACCAGGGCTGCGGCGGGTCTGG


 


CCCCGCGGGGAGGTAGCCCAGGGGCTGGAGAGGAGGAAGTTGGGGTGGGGGT


 


CCTGCAGGAGGAGTGCAGGCTTTGGGAGGTCTGGGGAGAGACTAGCGACTGC


 


ATCCTGTCGCAGGCGTGCTCTTCAGCCTGTTTGAGGCTTTATCGCCTCATAGGT


 


CCCGCGGGGCGGGACAGAGCAGCGACTGCCAGTTACTCACTGACCCAGGCCA


 


GTTTGATCAGGCACAGCTATTCGGTTTTTTTGGAAGCTCTCCCAGGGCCCAATA


 


GTCAAGGTTGTCGAGGATTACTTATTTCTTCCCCCAAATCCTCGCGATTTTGTT


 


ACAAAGACAGGTTTAAATGAAATTCTGCCATACCTCCAACCATTTATTAGCAA


 


GCACGCACGCATTCAAATTAAACGTCCTGCCTGTGAGCTTGACAACGTGCAAG


 


CTCCCCCTCTTTTTAGATGTTCTGTTCACATTCAGTATGAAAGGTTCCTTTTGTC


 


CTAGAGGTGGGATGAGGTTTACCTTACAGATGCCCAGGTCAGGCAACCAGAAT


 


GTTATTTCACAGCTGCACATGGTTAGGAATTAAGCCTGGAATGTCGCCCCCTAA


 


CGATATGGTCCCCAGGCCAGTTACTCCAGGCAAGTCTGGAGCATCTCTACATTT


 


TAGCCAGTCTCCAGAGCTAGAGTACCACCAATTTCACTACCATTTTCCATTCTT


 


TTTTTTTTTTTTTTTCTTTTTGCCATTTCTTGGGCCGCTCCGGCGGCATATGGAG


 


GTTCCCAGGCTAGGGGTCCAATCGGAGCTGTAGCTGCCGGCCTACACCATAGC


 


CACAGCAACTCAGGATCCAAGCCGCGTCTGCAACCTACACCACAGCTCACGGC


 


AACGCCAGATCGTTAACCCACTGAGCAAGGCCAGGGATCGAACCCGCAACCTC


 


ATGGTTCCTAGTCGGATTCATTAACCACTGAGCCATGACGGGAAATCCCCATT


 


GTCCATTCTTAACATCTGGTATGCTGATGGATAGGTCAGTAACTTTTACCCATG


 


AAAACAGGTTTAATATTCAGTATGCTAATTAGCACATCCTTGGGCCTTGACCAG


 


AGATGAAGGTTAATTGAAGCATTCTAGAAGCTACCTCTTGGAGTGCTGATTTCC


 


AGCGTAGCTTAAGTAGGAGACCCCCGACCGCTCTTTTGAGGAGTGACTCAGAT


 


ATATTTGCACAAACATACCACACTGTGTGATTTTAAAGTAAATCAGTGATATGA


 


AAACAGACCCTTGGGCAGGCAGTGGGTGTGAAGACTAGCTGCTGTGTGTGTGT


 


GAAATCTTTGAAGTTACTGCTTTGGGGAAATGCGTCTGCTTTCTGGCTCTAAGC


 


TCTTTTAATTTATCACTCTTTAATGAGGAAATGCTGGACCTCCTCTCTAGCTGTG


 


CTAAAAACACGTGAGTCCCAGTGAAGGCCTTTCTGTTGCTTCGGGAGAATAAT


 


ATTTTTGGTTTTGTCTTGTTTTTCATTGAGAAGTCATTCCTGGAAGATGGTGTTT


 


GTACCTCAAAGGCCCTCTGGCGTCTCTGAACTCATCGAGGATGGTGGACGGGA


 


TTAAAGAATGGCTTCTTGGGAGTTCCCATTGTGGCGCAGTGGAAATGAACCCG


 


ACTAGGAACCATGAGGTGGCAGGTTCGATCCCTGGCTTCACTCAGTGGGTTAA


 


GGATCTGGTGTTGTCGTGAGCTGTGGTGTAGGTCACAGACATTGTTGTGCCTGT


 


GGCATAGGCCAGGAGCTACAGCTCTGATTAGACCCCATAGCTTGGGAACCTCT


 


GTATGCTGCTGGTGCGGCCCTAAAAAGACAAAAAGACCAAAAAGAAAAAAGA


 


AAAAAGGAAAAAAAACAAAGAATGGCTTCTTCGGGGCTCAGCCTTCATGCATC


 


AGAGTAGTGGCTTTGGAGAAAGAGTTTGCCTAAAGGAGTTCGTATTTATATTTT


 


TTATTGAATTTATTCTTTTATTATTTAGTAATTGTAGTTGTGAAATTGTCATTAG


 


TTTGTATATTAAGGAATACTCTCTCCTGCTGAGCAGTTTATAACTTTTCCTGTCC


 


TCTGTTCCCATGTGGGCCTGTCTTCCCAGTGAGGATGTACACTCAACAAGGTCA


 


GGAACTCAGCTTCTGTTCTCCAGGGTTGCCACATTGTCTCTTCGTGGTTTTTAAC


 


TTTTTGGGGAAGCTGACGTCCTTGGATGTGCTGTGGGCTGGCTTGTGTCCTCTC


 


CCTACCCCCATTCATCTTTTGAATCCCTAATCTCCGATGTGGCTGTATTTGGAG


 


AAAGGGCTTTTAGGAGGTGATGGAGGTTAAATGAGGTCATAAGGGTGGAGTCC


 


TAATTTTATAGGAGTGGTGGCCTTCTCTACACACACACACACACACACACACA


 


CACACACACTGAGGAATGCCGTGTAGGCACGCGGCATGAAAGTGGCTGCTGGT


 


AAGGAGAGGCCTCCCCAGAAACCAGCTTTGGTACCTTGATCTTGGACTTGAGC


 


TTCCAGAACCGTTAGAAATACATTTCTGGGTGTTTGCACTGTGGCACAGTGGGT


 


TAAAAACCTGACTGCAGTGGCTCAGATCTCCTGTAGCCGTGTGGGTTCCGTCCC


 


TGGCCCAGTGCAGTGGGTTAAGGATCCAGCCTTGCCACAGCTGTGGTGTAGGT


 


TGCAGCTGCAGCTTGGAGTCAATCCCTGCCCTGGGAACTTCCATATGCTGCAG


 


GGACAGCGGTAGGAAAAAACAAATAAACAAATAAATAAATAAATAAGGAGTT


 


CCCATTGTGGTACAACAGGGATGAATCCAACTAGGAACCATGAGGTTGCAGGT


 


TCGATCCCTGGCCTCGCTCAGTGGGTTAAGGATCCGACATTGCCATGAGCTGTG


 


GTGTGGGTCGAAGATGAGGCTCAGATCTGGTGTTGCTATGGCTGTGGTGTAGG


 


CTGGCAGCTGTATTTCAGATTCTACTCCTAGCCTGGGAATCTCCATGTACTGCA


 


CAGGGGCAGCCCTGAAAACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA


 


AAAAAAAAATTTAATTACTTAATTTCTCATTAAAGCCATCCAGGCTGATATTTT


 


GTTATTGAAGCCTGAGCAGAATAATACAGTATATCTGATAAGATCTATAGTCTC


 


CTGTGAAAAAAATGCACATAAACATGTTAACATCACATTTTGATAAAAGTTGA


 


GGTAGGGAGTTCCCGTTGTGGCTCAGAGGGTTAAGAACCTGACTAGTATCCAT


 


GAAGATGTGGGTTTCATCGCTGGTCTTGCTCAATGGGTTAAGGATCCAGCATTG


 


CTGTGAGCTGTGAGCTGTGGTATAGGTTGCAAATGTGGCTCGAATCTGACATG


 


ACTGTGTTATAGGCCTGCAGCTGCAGCTCTGATTTGACCACTACCCCACGAATG


 


TCCATATGCTGCAGGTGTGGTCCTAAAAAAAAAAAAAAGGTGAGGTGGGCCAT


 


GGACCTGCTTGATGCCTGTTCATGGTCCATCTGGGATCCAGTGCTGTGTTCAGG


 


GCAGGCTTTAAAGCATCTGTAAGGCCCAGAGGACAAGATGGGACCCATGGCCT


 


GAGATTGCAGCCCAGGGAGGGGTAGGACCCCTTCAGGAGGCAGCTTAATTTGA


 


TGTATTTTGGTCAAAGGAAAAGCCAAAGGCCCTGGAAAGGAGGCTTGGGGTGC


 


ATCCTGGTGGCAAATAGGGAACTCACCCAGGTTCCCTCCACTGGTGGGATGGG


 


GTGTCTTGATGGAACAGGATGCTGCCCAGAGGCTTAGTTTGGGAGCAAGGAGC


 


AGAGTCCAGGATCAGCCTCCTTATTGCTCTGACTCAAACTCTCTGGAAGACTTG


 


ATTAAGGAGAGTCCTGCCTGGGGACATACTCATTCTTTGGCCCTAAAGCCTGCC


 


TCAGGAGTGCCCTGAGGAGAGGCACCTTCTGAGGGCACATGGTCTGTCCGTGT


 


GTGGTCTGGTGACTGCCTTGTTTTTGAGAAATGAGGACCGCCATGTATACATCG


 


AGGGCATGCGTGTTGGTTGTAATTAGGATAATTCAATTTCTTTTTTTCTTTTTTA


 


TGAGAGTGGTAATGTTTATTTCAGGTTCTCTCTCTCTTTTTGTCTTTTTTAAAAA


 


AAATTTTTTTTTCTTTTTTTGCCACACCCGCAGCATGTGGAAGTTCCAGGGCCA


 


GGGATTGAACCTGCGCCACACAGCAGTGATCAGAACCACAGTGGGGAAAATG


 


CAGAAGCCGAGTTCCCATTGTGACACATCGGAAATGAATCTGACTAGTAACCA


 


TGAGGTTCCGGGTTCGATCCCTGGCCTTGCTCCGTGGGTTAAGGATCCGGCATT


 


GCCATGGGCTGTGGTGTAGGTCACAGACGCGGCTCGGATCTTTCGTTGCCGTG


 


GCTGTGGTGTAGGCTGGCAGTTGCAGCTCTGATTAGATCCCTAGCCTGAGAAC


 


CTCCATATACTGTGGGTGTGGCTCTAAAAAGCAAAAAACCAAAAACAAAAACA


 


AAAAGAAGATGCAGGAGCCTTAACCCACTAGGCTATCAGAGAACTCCCTCGAT


 


TTTTTTCTGATGGGTGATTGTTTAACATCATCTCCCTCACAGCCCAGCTTGACTG


 


CCTTTGCCTTCTGCCTTCATCAAGGGCGGTCATGGGCAGTGTTTACATATATTC


 


ATCAAAAATGCACATGAAAGTCATTTTTGGTACATGATTTGACTTTTTTTTTTTG


 


GTCTTTTTTAGGGCTGTACCCATGGCATGCGAAGGTTCCCAGGCTAGGGGCTA


 


ATTGGAGCTGCAGCTGCCAGCCTACACCACAGCTCACGGCAACGCCGGATCCT


 


TAACCCACTGAGTAAGGCCAGGGATCGAACCCACAACCCCTGGTTCCTAGTCG


 


GATTCATTTTCACCACGATGGGAACTCCCATTTTTGGTGTATGATTTGAAAAGA


 


CTTTCAAAATAACCTGCTTGTTTATGGGTTTGTTTTTGTTTTTTTAAGTAAAATG


 


AACTTTCCAACAGCATTCATGTATTAACCGCAAAAGAGCTGGAAAAGCATTAT


 


GTAGAAGAATAATTTCGTTTTAATTAGCTTTAAGCTTATTAAACTTATGATCAG


 


TGTTTAGAAATCTTATAATGAAGATGGTTATTCATGGTTAGACAGCAATGCATT


 


TTTTTTTTTTTTTTTTTGTCTTTTTGCCTTTTTCTAGGGCTGCTCCCGTGGCATGT


 


GGAGGCTCCCAGGCTAGGGGTCGAATCAGAGCTGTAGCTGTCGGCCTACACCA


 


TAGCCACAGCAATGCCAGATCCGAGCCATGTCTGCGACCTACACCACAGCTCA


 


TGGCAACGCCAGAACCTTAACCCACGGAGCAAGGCCAGGGATTGAACCCGCA


 


ACCTCATGGTTCCTAGTCGGATTCATTAACCACTGAGCCACGATGGGAACTCC


 


AACAATGCATTTTTTTTTTAATTTAGTTTTTAAATTTTTTGGTTGCACCCAAGGC


 


CTTGGAAGTTCCCAGGCCAGAGACTGAATCCGAGCTGCAGGTACGGCAACACC


 


GGATCCTTTAACCCACTGTGTCAGGTCAGGGATGGAACCTGTGCCAATGCAGG


 


GGTCCAAGACACTGCAGTTGGATTCTGAACCCACTGTGCCACAGCGAGAGTCC


 


AAGCAATTCATTTTTTAAAAAATTCTTTTTTTTCCTTTTTCTTTTGGACCCCCTTC


 


ACCTGATTCTCCCACCTCCTACCCCCCGACCTTTGTTAATCAGCAATATGTTCT


 


CAGTATCCGTGAGGTTGAATTTTTGCTTTTTCACATTTCATAGAAAAGTGAGAT


 


CATGCAGTATTTGTCTGTGTCTGACTATTTCACTTAGCTATGCCTTTGGGCTCCG


 


TTCATGTCACAAATGGCAAGAGTTCATTCTTTTTTACAGCTGACTAATATTCCA


 


TTGTGCATATGTATACCACAATGTCTTTATCCATTTATCTCTTGATAGACACTG


 


AGGTTGTTTCCATGTCTTGGCTCTTATAAATAATGCTGCAGCAAACACGAGGGC


 


ACAGATACCTTTTCCAGTGAGTGTTTTTGTTTTCTTCAGGTAAATAGCCAGAAG


 


TGAAATTGTTGGATCTTGTAGTTCTGTTTTTAATGTTTTGAGGAACCTCAGCCTC


 


CTGTACTGTCTTCCATAGTGGCTGCACCAATTTACATTCCCACCAACAGTGCAC


 


AAGCGTTCCCATTTCTCCACATTCTTTTCAACATTTGTTCTTTGCTTTCTTGTTG


 


ATCATAGCCGTAATGGGCATGATATCTCAATGTGGTTTTGACTTGCATGTCCCT


 


GATGATTAGTGATGTGGAGCATTTTTTCGTGTACCTTTTGGCCATACAATATGT


 


CTTGGGGAAAAAAGTCTATTCAGATCTGCCCATTAAAAAAAAAAAATCTCTTT


 


TTTTTTTTTTTTTTTTTTTTGCTGTTAAATTTGTATGAGTTCTTCCCCACCCCTTT


 


CATTTTATGCCCAGACTTGCAGCATGTGGAAGGTCTCATGCTTGGGTCAAATTG


 


GGACTGCAGCTATGGCCTATGCAACACCAACACCAGATCAGAGCCACAACTGA


 


GACCTATGCCAAAGCTTGCGGCAACTCTGGATCGGTAACCCACTGATGCAGGC


 


CAGGGATTGAATGTACATCTTCTCAAACATTATGTTAGGTTCTTTATATATTTTA


 


GATATTAGCCATGCATCAGATACATGATTTGCAAATATTTTCTCCTCTTAGTAG


 


GTTTTTTCATTTTGTTAATGTTTCCTTTGCTATGAAGAGCTTTTTAGTTTGATGT


 


AGTCCCACTGTTTATTTTTGCTTTTGCTTTTGGTGCCATATTCAAAATAGCATCA


 


CCAAGACCTGCATCAGAGAGATTACTGCCTATGTTTTCTTATAGAAATTTTATG


 


GTTTCAGGTCTTACATTTAAGGCTTTAATCTGTTTTGAGTTATTTTTTGTATGCA


 


GTGTGGTGTGGTAAGACAGTGCTCAGGTTTCATTGTTTTGCATGTGGTTGTCCA


 


GTTTTCCCAACACAATTTATTGAAGAGACTTTCTTCATTGTATAGTCTTGACTTC


 


TTTACCATAAATTGATTGATGCTATATGTGTGGGTTTATTTCTGGGCCTTGTATT


 


TTTTTCTATTGATCTATCTATGTTTTTATGCCAGTGCCTTAATGTTTTTTTTGCTT


 


TTTCTTTAGGGCCACACCATGACACATGGAAGGAAGTTCCCAGGCTAAGGGTT


 


GAATTGGAGCTTCAGCTGCCGGCCTACACCACAGCCACAACACAGGATCTGAG


 


CTGCATCTGCGACTTGCACCACAGCTCACAGTAATGCCAGATCCCCAACCCAC


 


TGAGTGAGGCCACAGATTGAACCCGAGTCCTCATGGATGCTAGTCAGATTCAT


 


TATTGCTGCACCACAATGGGAACTCCCATAATGTTTTAATTACTTTAGCTTTGT


 


AATACATTTTGTAATCAGGGAGCATGATGTTTCCAATTCTTTTCTTTTTCAAGGT


 


TGCTTAAAAAGAAATCTTTTGTGGTTCTATACTAATTTTAGGATTATTTGTTCTA


 


TTTTTATGAAAAATGTCATTGGAATTTTGATATGGTTTGCATTGAACCTGTTGA


 


TTGCTTTGGGTAATATGGACATTTAATAATATTGATTCTTTCAACTCATGAGCTT


 


GGATTAGCTTTCCATAACTTTCCATTTATTCGTGTGATGGTAGTTTTTCTGTTAA


 


CGTCTTGTATTTTTCAATATGCAAGTCTTTCACGTCTTTGGTTAAACTTATTTAT


 


AGATATTCTTTTTGATGCAGTTGTGAATGGGATTGTTTCTAAATTTCTCTTTCTG


 


ATGGTTTGTTTTAAGAATGTAGAAATGGGAGTTCCCATCGTGGCACAGTGGTTA


 


ACGAATCCGACTAGGAACCATGAGGTTGTGGGTTCAATCCCTGGCCTTGCTCA


 


GTGGGTTAAGGATCTGGCGTTGTCGTGAGCTGTGGTGTAGGTTGCAGATGCGG


 


CTCGGATCCCATGTTGCTGTGGCTCTGGCGTAGGCCAGTGGCTACAGCTCCGAT


 


TGGACCCCTAGCCTGGGAATCTCCATATGCCGTGGAAGCAGCCCTAGAAGAGG


 


CAAAAAGACAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATGTAGAAATGCA


 


ATGGACTTTTTGTGTTTATGTATCCTGCAACTTTACTGAATTCTTGTATTAGTTC


 


TAACAGGTTTTGATGGAGTCTTTAGAGTTTTCTCTGTATGGTGTCATGTTGTGTT


 


TGTAAATAGTGTCAGTTTTATTTCCTCCTTTCTACTTTGGATGCCTATTATTTCT


 


TTTTCTTGCCTAATTGCTCTGGCTAGGACTTATAGTACTATGTTGAATAAAAGG


 


GCAAGAGTGGGCAGTCTTGTCTTGTTCCTGATATTAGAGGAAGTCTTCATCTTT


 


TGATCATTGATTATGATGTTTGCTGAGGGCTCACATATGCCCTTTATTATGTTG


 


AGGTATGTTCCCTCTATACCTGATTTGTTGAGAGTTTTGTTTTTTTTTTTTTTTAA


 


TCATAAATGGATGTTGAGTTTTGTTCAATGCTTTTTATGCATTTCTTGAGATGAT


 


TATATCATTTTTTATTCTTCATTTTGTTTATGTGATATATCACATTGGCTGATTTG


 


TGGATTTTGAACCATCTTTATGTCTCCAGAATAATAAATCCCACTTGATCATGA


 


TGTATGATCTTTTTAATGTATTGTTGAATTCTGTTTGCTGGTATTTAGTTGAAGA


 


GTTTTGCATCTATGTTCATCAGGGATATTGACCTGTAGTTTTATTTATTCTTTTT


 


TATGGTGTCCTTGTCTGGCTTTGGTACCAGGGTATTGCTAGCCTCATAAAATGA


 


GTTTTTTGAATGTTTGTAGAATTCACTAGCAAAGCCATCTGGTCCAAGACTGTT


 


TGTTGGGGTTTTTTAATTACTGATAGTAGAAAGGATATTCAATATCCTTACTAG


 


TTATCAGTCTGTTCAGGTATTCTGTTTCATCATGGTTCAATCATGGTAGGTTGTA


 


TGTTTCTAAGAACTTACCCATTTCTTCTAGGTTGTCCAGTTTGTCGATTTATAAA


 


TGTTCACAGAAGTTTCTTACCTTTCTTTTCTGTTGTAATGGTTGTAATAAATCTT


 


CTTCAATTTCTGATTTTATTTATTTGAGTCCTATCTCTCTCTCTATTTTTGTTTTT


 


TTGGTGAGTCTAGCAAAGGCTTGTCAATTTTGCTTACCTTTTCAAAGAATAAGT


 


TCTTAGTTTCATTGATTTTCTCTGTTATCTTTTTAGGTTCTAGTTCACTTATTTCT


 


TCCCTAATCTTAGTTATTTTCTTCCTTCTACTAATTTTGGGCTTTGTATGTTCTTC


 


TTTTTCTAGTTTCTTTCTTTCTTTCTTTTTCTTTTTAGAGCTGCACCTGCGAAAGT


 


TGCTGGGCTAGGGGTTGAATTGGAGCTGCAGCTGTTGGCCTGCGACACAGCCA


 


CAGCAACACCAGATTTGAGCTGCATCTGTGACCTACACCACTGGATCCCGCTG


 


AGCAAGGCCAGGGGTCGAACCCACCTCCTCACGGACACTATTGTCGGGTTCTT


 


AACCTGCTGTGCCACAACAGGAATTCCTTTATCTCAGTTACTGAAGTGTAAAGT


 


TAGGTTGATTATTTGAGATTTTTCTTATTTCTTGAGGTAAGCACTGAACCCTTGA


 


ATTTCCCCTTTAGAACTGATCTTGCTGCATCCTGAAAATTTTGGTATGTTTTTCT


 


TTTGTCTCAAGGTATTCTTAAATTTCTTTTTGATTTTTTCTTTGACCCATTGGTTG


 


TTTAATAGCCTGTAACTTAATTTTCACATTTGTGAATTTTCTAGTTTTCTTCCTG


 


TAATTAATTTTTAGCTTCATACCATTGCAATTGGAAAAGATGCTTTATATGATT


 


TCAGTCTTCTTAAATGCGTTAAACCTTGCTTTGTGGCTTAATATGTGGTCTATCT


 


TGGAAATTGTTCTATGTGCACTTGAGAAGAATATGTATTCTGTTGCCATTGCAT


 


GGAATGTTCTGCATATATTTGTGAAGTTCATCTGGTCTAATATGTCATTTAAGT


 


CCAATATTTCCATACTGATATTCTATCTGAAATGTTCTATCCATGATGTAAGTA


 


GAATATTAAAGTCCCCTGCTTTTGCTATCTGTTTCTCCTTTTAAGTCTGTTAATA


 


TTTGCTTTATATATTTAGATGTTTCTGTGTTGGGTGTATAAATATTTACAAATGT


 


TGTATCTTCTTATTGGATAGATCCCTTCATCATTGTATAATATGGTAAGGTCTCT


 


CTCTCTCTCTCTTTTTTTTTTTTTTCTTTTTAGGGCTGTACCTGCAGCATGTGGAG


 


GTTCCTAGGCTAGGGGTCAGTGTGGAATTGTAGCTGCCAGCCTATACCACAGC


 


CACAGCAATGCGGAATCCAAGCTGCATTTGTGACCTATACCATAGTTCACAGG


 


AGTGCTGGATCCTTAACCCTCTGAACGAGGCCAGGGATCGAACCTGTGTCCTC


 


ATGGATGCTATTCAGATTTGTTTCCGCTGAGCCATGATGGGAACCCCATGGTAA


 


TGTCTCTTATTACTGTCTTTGTTGTTGTTTTTACTTTTTTATGGGCTGCACACGTG


 


GCATATGAAAGTTCCTAGGCTAGCAATTGAAAGTGAGCTGAGGCTGAGGCCTA


 


TGCCACAGCCATGGCAACACTGGATCCAAGCTGCATCTGTGACCTACTCTGCA


 


GCTTGTGGCAATGCCGGATCCTTAACCTACTGAGTGAGGCCAGGGATCAAACC


 


TGCATCCTCACAGAGACTGTTGGGTCCTTAACCTGCTGAGCCACAGTGGGAAC


 


TCCCAGTCTTTGTTTTAAAGTCTATTTTGTCTAAGTATAGCTACTCTAGCTTTTT


 


TTTTTGGTTTCCATTTAGAATTAGGGTAATTAGAAGTTCCCATTGTGGCTCAGC


 


AGATTACAAACACAACTAGGATCCTTGAGGATGTGGGTTTGATCCTTGCCCTCC


 


CTCAGTGGACCTGGCATTTTCGTGAGCTATGGTGTAGGTCACAGATGTGACTTG


 


GATCCTGTGTTGCTGTGGCTGTGGCTGTCTGGTTTGCTTTAAATGTCACCTTTCT


 


CTTTAAGTTGCCTTTCTGTGTTTGTTAATTTGGAAAGCTGTGGAGCAAGCCTCT


 


GTGGGTGGTAATAACCCACCTTTCAACCCAGCAGTTATAGTAGGTTGAGAAGT


 


AATGAAACTTTTTTTTTCCCCTGGCCTAAGCTTGAATGGTTTCCTCAAGGATTC


 


CATGGAAGCAAACTTGAATATCTAGGTGGACTAACTTAAGTGATTGGAATTGA


 


ATGGGTCACTGAGAGCCTGGACCAAAGGGTAGTTGGATTGCAATGAAAGAAG


 


AGAGTAAGGGAAAATGATGGAAGCATTTCCCAAGGAGACCCGGTCACCTACTC


 


TTCTTTGGCTACTCTGAGTCATTTAGACTCTTCCCCCAGGGGTAGGACATCATT


 


CTCCTTTTAAATGGCTTCTGATGGCTTGGAGATGGTCCATTTTTTAGTGGTTCTT


 


ATCCTGTTTTCAGTGGATGCCTCACTTGCAGAAGCAAAAGCCAGTAGCTCATG


 


AATCAATTCTCTTCTAAAAGTGAAACTCTCATTTATTTTCTCTTAAAAGGAAGC


 


CTAAAATGATCAGATAACTCAGCTTCCTTGGGGCAAGGAATACTCTGAAAAAA


 


TAATATATTTCTGATTTTTTTTTTTTTGGCAGATCACATGAAAGGATTAAGAGC


 


CTCTACCTTGAAAAGAAGGTTGGAGTGCAGGTAATACATGTTGCTACTTATTTG


 


TGCGTGTGTTTTTGTCTTTTTAGGGCCACACCTACGGCGTATGGAAGTTCCCAG


 


GCTAGGAGTCAAATCAGAATTGTAGCTGCTGGCCAATGCCACAGCCGTGCCTA


 


CCTGGGATCCGAGCCATGTCTGCGACCTACACCACAGCTCACCGCAATGCTGG


 


GTCTTTAACCCACTGAGCGAGGCCAGGGATCAAACCCACATCCTCGTGGATAC


 


TAGTCGGGTTCGTTACTGCTGAGCCACCAGGAGAACTCCTATGTGTGCGTTTTT


 


AAGTAAAATTTTACTTGAGCTGTAACTTCTACACAGAAATGTACAGACATCAT


 


AAGGGAACAGTCTGACAAACATTTGTAAGTGAACAGATCTGTGTAACTTCTGC


 


CCAGCTCAAGACGTAGGACAGAACCAAAATCCTGGAACAGACCCCGTCCCTCC


 


CCCAGTCAGTCTTCCCTGCCCACGGGCAACCTCTTTTCTGACGTCTGTTGTCAT


 


GTTTAACTTGGGCTGTTTTTGAACGCTATTTAAATTGTTTCATACTGAATAGACT


 


CTCCTACTTTTTTTCTTTTACTTAGCAGTATATGTGAATGATTCATCTGTGCTAT


 


TGGGCACAGCAATGGTCTGTCCATTCTGTCCATTTTCTTTCTTTCTTTCTTTTTCC


 


TTTTCTTTTCTTTTCAGGGCCACTTCCATGGCATATGGAAGTTCCCAGGCTAGG


 


GGTCGAATCGGGGCCGCAGCTGCCAGCCTATGCCACAGCCACAGCACTGTGGG


 


ATCTCAGCCATGTCCAAGACCTACACCACAGCTCACAGCAATGCCGGATCCTC


 


AACCCACTGAGGGAGGCCCGGGATTGAACCTCCATCCTCATGGATTCTAGTCA


 


GGTTCATAAGCTGGTGAGCCACAGTGGGAACTCCCACGTTTGTTTCTGAAAGG


 


GTGTGTATGTGTGTAAAGGGTTGTTTCCTTATAAAGAATTAAATAGCGTTGTTG


 


GTTCTCTTCTTAAAGCTGAGCCTTCGCATTCTTTTCCTTCAGCCATTGCTTAGAT


 


TCAAAATTATCAAGATATATCCAGTTGCCTCTTGAACCATGTGAGGGTTCGGGG


 


TGCTGACTTTCGTCGTGGTTGAAAATTGATGTGTAACATTACAGTCGGCCCTCC


 


CTATCCCCGGGTCTGCATCCATGGATTCAACCAACCGTGGACTGTGTCGTGCCA


 


TAGCACTCATTTAGTGAAAACAATTTCTCACATAAGTGGATTGTTGCTGTCGAA


 


TCCTGTGTTGTTCAGGGATCAGCTGGGTGTGGAAAGGAGTGTCTTGGCGTTTTG


 


ACTCTTTCACCTTTGACTTAGCAGCAGGCTTCTTTGCTAAGTGATATCAGCAGT


 


CTGTGTTTATGATAATGGTAACAAATACACACATGCACACACACACACACACA


 


CACACACACACACACTAGATGTTGATGATATGGTAATGAGTAAAACAAAAATC


 


CCTCTCTTCTTGGAGCTGACAGTCTCTTTAGAGCCTCTTTTGGAAAGACACTAC


 


TATTGCTTCTATTTTACAGATGAGGAAACTCAGGAACCAGAGAGGTTAAATTA


 


GTTGCCCAAGGTCACACAGCTAGTGGCAGGACCAGGATTCCCACCAGGTCTGT


 


CTGATTTTAGAGCCTATGTATTAGTTGTCCATTGCTGCGTAACAAATTATCCCC


 


TGAAGTGACTTAAAAACAACACTCGTCATGTATTGTTATCTGTTGCTGTGCCTC


 


AGGGATTCTGACAGGGCACAGTGGGAGCGGCTGGTTTCTGCCTGACTGGGTAT


 


CATTTGGGGGCGACCTTACCTAAAGGCTCGTTCATTCAATGTCTGATGCCTGGA


 


CTGGGGACACTTAAGTAGTTGGGACATTTTGGGTATCTCTGTCTCTAAACAAAT


 


CTTTGCAGCATGGTGGTTTCAGGATAGCTAGCGTCCAAAGTCCTGCAGTGCTAT


 


GTCCGTCCCAGTCTGTTGACCAAGGCAGTTACAAGGGTCTGTCCAGGTTCAAG


 


GGAGGTGAATGGAGACGCCTCCCGTCAAGGGAAAATGTCCATTCCCATGTGGT


 


TGGAGTGCACGTCAGCGTGGCCAGCTTTGGAAAGTATAATCTGCCGTAGATTG


 


TACCCCTAACCTTTCAGCTCTAGGGGTGAATTTTTCAAGCTTGTCATGAAATTG


 


ATTTCTGCCCAAAAGACACAGACAGAAGCGACTTGCACGAAGTAAATCTCAAG


 


CAGACTTTGGCTCCCCCTTCCTGTTTAGCAGAGGTAGTTTATCATTTGGTGGTG


 


TCTTTCCAGGCATAGCTGATCCTGTAACAACCATGTCTTTAAGATGAAAATAAA


 


CAATGAGGTCCTGCTGTACAGCACGGGGAACTATATTCAGTCTCCTGGGATAG


 


ACCACGATGGAAAAGAATATTTTTAAAAAGTGTGTGTGTATGTATGACCGAGT


 


CACTTTGCTGTCCAGCAGAATTGGCATAACAGTGTAAACCAACTATGCTTTAAT


 


ACTAATAATAACAAAAGATGAGAATGGTGAAGACCAAGCTTTGAGGAGGTCA


 


GAGAACATAATATTCATTCTCATCTGCTTCTTCCCCTTCACTTTATTTTATTTTTT


 


TAATTTATTTTTTTAGGGCCACACCTGAGGCATTTAGAAATTCCCAGGCTAGGG


 


GTCGAATCAGAGCCTCAGCTGCCGGCCCACAGCACAGCCACAGCAACGCCAG


 


ATCTGAGCCGCATCTGTGACCTGCACCACAGGTCACGGCAATGCCGAATCCCT


 


AACCCACTGAGCGAGGCCAGGGTTTGAACCTGCATCCTCATGGATCCTAGTCG


 


GGCGCGTTACTGCTGAGCCACGATGGGAACTCCCCTTCCCTTTACAGTGATGG


 


GGCGGAGGTGGTCATTGGAGGTTAGGCCTATTGGGAACCATTCTAACACTTTT


 


GGTGCCAGCTCCGTTTCTGAATTATTCAGGGTATCTGATACATGTTGAAACAGG


 


AGATTTCCTAGTCACCCAGATGACTCATTTTGGATGGTACTAAAATGACTTTTT


 


TTTTTTTTTAATCTCTCTTTTCAGCTTGAAGGTCTCCTTATACTGTTCACCTGTTA


 


CTAGAGAATTGTTATTAACCAACCCGAGGTACAGATTTTGGGAGAAACGAATT


 


GTAAGTTTTATTTTTTTCGAATGCCACTAATTCTTTTTTTCAAGTGGGAACTACT


 


TTTCGAGGGAGATGGAACAAATGTATAAATATATTTATCTGCAGTATTTATGGT


 


GTCGCTATTCACTCTTTTGTCATTCCTTAATTGTGGAAGAGCTTGGCTTTTTATT


 


GTTGTCACTTTGTCTTTCTTGTCCTTGAACACGTTGACCTAGATTCTTAGTTTTG


 


TCTCGTCTCAAAGACCCTCTTGCCCAGCTGCATTTTAATAGTTTAAAGCATAAA


 


ATGTTCAGTGACATAATCGATAGATCCTTTTATTTAGACCATGGGTTAGGTGAG


 


GAAAAATGAAGAGGACTTAGGAGTTATAATAAGAAAGGAATAATAAGCTGAA


 


TAAAAACAGGATGGATAATTTGAATCTGACAGAATAAAGTGGATCTCTGCAGT


 


GGGAGGCGGATAGTAGAGGAGAGAAGACCATATGGCAGGTAGAAAATGTGAA


 


GATGGCAGAAAAATTTCTGTTGCAACAGTAGTTGCTATCCACATGAATGGATTT


 


CATTCAATTTGTGATCCAAAGACGAAGTCGTAGTTTAGGTTTAAAAAAAAAAA


 


ACAACCCAGCCTGGAGTTCCCGTCGTGGCTCAGTGGTTAACAAATCCGACTAG


 


GAACCGTGAGGTTGCGGGTTCGACCCCTGGCCTTGCTCAGTGGGTTAAGGATC


 


CGGCGTTGCCGTGAACTGTGGTGTAGGTTGCAGATGCGGCTTGGACCTGGCAT


 


GGATGTGGCTGTGGTGTAGGCTGGTGGCTACAGCTCCAATTTGACCCCTAGCCT


 


GGGAACCTCCATATGCCGCGGGAGTGGCCCTAGAAAAAGGCAAAAAGACAAA


 


AGCAAACAAACAAAAAACCTAGCCTTAGTCTATAAGAGACGTAGATGACATTA


 


AAGGAAAAACACAGGTCGAAATTGGGGGAAAAAAAAAAGAGAAAAGATATA


 


CTGAACAATTAGAAGCTAAAAATGCCAAAGTGGCAACTTTTATATCATGTAAA


 


GAGCTGAAGGAAAAAAAGAAAATCTAAATTTTCAAGAGGGTAAAAGAAGATC


 


AAGAAGATGTGAACACACACACACGTACTCACAACTGACAAAATAACCTCAA


 


ACTGGACTCTGTCGTAGTTGGTCATCTAGTGCTTGATGGGAGAAAGAGAACCG


 


TCATTCGTAGGTAGAATTCTGCTTCATTATGTGGCTTAAACTGCCAGTAATTGG


 


GGCAGCTGGCAGAGAAGTCTGAACAAGCATCTCAAAGAGTCTTTTTGATGTCT


 


GACTGCCAATAACTTACAGCTCCCACCTCCAGCTTCCCTTTCTGCCCCACATCT


 


GGGCAAACTGATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA


 


AAAAGCCTGTCCTTTCCTGGGTGCCTACATGGAGTTCAAACATGAAAGCTCAG


 


GGCCACCTGTAACCACAGTGAAAACCAGGGCCCCTTGTTCCTATTTTCTCAAGC


 


TACCTGGGCCACTTCCTCCCAGCCTGGGTACCAGCCTCCGCTCCCCAGAACGTC


 


CCAGGATATGGGTAGTGCAGTGTTTTCATACCCTCTTGGGTCATGTTTGGTGGA


 


ATTTTCAGTCTCAGCTTCTGATCTGTCCCTTAGGTGGGACCCGACAGAGGCCAT


 


CCCTATGGAGAGGGACCCAACAGCAAGGCTGCTAAGTAACCTTGGCCTTGGTG


 


TTAGGCCTGCAGTTGCTGTGGGTCATTCCGGCCAGCAGTCAGGAAGGGAAGCT


 


GAGGAAGAAGGGGAGAAGAGCAGGGATAGGCTGGAATCCACGGGGACAAGT


 


GGGGACCCATGCCTGCCTCTCACTCCTCCCAACCTTGGTAATGCTAACGCTGCT


 


GTTCCCTTGGCCACTGAGTCACACATACGAGCCCCAGGGCTCCGAGAAGCTGA


 


GCGGGAATATCCCGCAGAAGATGGAGCTGCGCTGTCCTGCCCGTGTCAGTGAA


 


CTGAGCCTGCAGATCAGCAACACAGGAGTTGTTGTCATTCCTGGTGCCTTGCA


 


GAGCATTGAAAAATGCTGCTGGCTGACGTCTGCCATCCGAGTCTTACCCAGGT


 


GTTTCTTGGGGCTACTGCTGACCCAGACGTATACACAGCAGGAGCTTACAGGA


 


TAGTCCAGGCTCCGCTGAGTTGATGTAATTCAAGGCCACCTCGCAATCTAAGG


 


GGGAAGGGGGCAGCTTGATAGAAGCAGGTGGAGAGCTTACCAGACACTCTCA


 


GAAACTGAACACGAGGGACGTGGGAAGTAAGTAAATAGCCAGAAGGCTTAAA


 


TGACGTAATGAATGCATGTGAACTGATTGAAACACAGAGCTTTAAACCCAGCG


 


AAGGAAGGAAGATCTTTGGGTGCACGTGGAACATTGACCAAAATCAGCAAGT


 


GCTTTGTCACTGACGCCTTTCAAGAAATCCTAGTGATTTGTTCTTTGACCGTAA


 


TGCAGTGATATTGGAAATCAGAAATAAAAGGAGAGTTAAATTCATGAATGGGA


 


ACCTTTAGTTCCCATTCATGTGGGAACTAAAGGTTCACCACAAACAACTCTCAG


 


GTTAAAGAGGAAGTTGTAAAGAAAAATATACAATACTTAGGACTGAGGAACA


 


ATAAAAAGAGGACATTCAAAATGTGTGGAGCACAGCTCAATTAATACTTAAAA


 


GGATGGAGTTCCCATCGTGGCACAGTGGAAATGAATCCAACTAGGGACCATGA


 


GGTTGCGTGTTCGATCCCCGGCCTAGCTCTGTGGGTTGGTGATCCGGCATTGCT


 


GTGAGCTGTGGTGTAGGTTGCAGATGCAGCTCGGATCTGGCATTGCTGTGGCT


 


GTGGCGTAGGCCGGCAACAACAACTCTGATTAGAACCCTAGCCTGGGAACCTC


 


CATATGCTGCAGGTGCAGCCCTAAAAAGACAAAACCCCAAAAAAAAAAAAAA


 


AAATTAAAGGGAAATTGACAGCATTAAATGCATTTCTTAAAAAGCAAAGACGA


 


ATTAAATGGAAGAATTCTTTGACTTATTAACAAGGAAAAGAAGCAACAAAGCA


 


AATCTAAGGACATGTAAAAGAAGGAAATAATAAAGAGAAAATGACAGATTAG


 


TGATATAGAGACTAACTATCACAGAAAATTAACAAAAGTAAAAACTGGCCTTT


 


AGAGAAATAGAGATAATTTTCTGCCAAGATTGGTGAAAAAAGATGTATATAAA


 


AATACAGAATGATAGGTGTTCCCATTGTGGCACAGCGGAAACGAAGCCAACTA


 


GGAACCATGGGGTTGTGGGTTTGATCCCTGGCCTTGCTCAGTGGGTTAAGGATC


 


TGGCATTGCCATGAGCTGTGGTATAGGTCACAGATGCAGCTCGGATCCTGTGTT


 


GCTGTGGTGTAGGCTGCCAGCTGTAGCTCCGATTTGACCCCTAGCCTGGGAAC


 


CTCCATATACTGCAGGTATGGCCCTCAAAAGCAAAACAAACAAACAAACCCAA


 


AACAGAATGATAAAAAGGAAAATAACTATTAATAAGAGCAATGCACAATGCC


 


TTATTTTCAAAATTAATGTGGTAGTGTATTTGATAATTTAGACACAGTGGATAA


 


ATACCTGGGAAAGTAGGAAAATACCAAATTAAGGCAAGAAAAGTAGGAAACT


 


TGAATAAAGAAATAGCCATCAGGCATTCCCATTATGACTCAGTGGGTTTAAGA


 


ACCCATATAGTCCCTGTGAGGCCTTTGTTAGTGNNGTAAGAATCTGGCATGGCT


 


GTACACTAGCATAGGTTGTAGATGCAGCTCGGATCCAGCATTGCTGTGGCTGT


 


GGCTGTGGCCTGCAGCTGCAGCTCTGATTTGACCCCTATCCTGGGAACTTCTAT


 


ATGCCACAGGTGGAGCTGCAAAAATGAAAAAAAAATTTTTTTTAATTTAAAAA


 


TTTTAAAACAGTTTTGAAAGATCTCTTCTTCAAAAAAGCCCCAGGTCCAGATGG


 


TTTTATGGGCACTTTGGCCACATGTTCAGAGAACAAGTAATTCTTGTTTTAGGA


 


GAGAATAAAAAGATAAGCCAGTCAACCTCACTGGCAAACCGGATAATGGTAG


 


CACATGGGGAAGAAAGTCCATTTCATTCATGAGCATGTGTGAACATCCAAATT


 


AGGGTAATAGCTAACTGAACCTAACAGTGTTAAAAAAAAGTGCATCACAAATA


 


GAGTGTTTATTCTAGAAATGCATGAACATTTTTATACCTGAACCACAGTCACTG


 


TGAGTTTTATGGTATCAGTGGACTAAAGAGATGAACAATTATGTTCATTCATCA


 


GCTACTTACTGAATGCCTCTTTGTGCTGGTGACTGTTCCAGTAAATTGTCCAAG


 


GACCCTTGGACCGTTCTGCAGCAGCTTGAATCTGTAAATTTGGCCAGTGTGTCA


 


GGACTGATGGCAGACTGTTCTGCTGCAGAAAACCTAAACACATAGAACACCTG


 


ACTACCTGTCGTACAAAGACTAAGGTCAAAAGGAAAATGGGATGGGAGCAGG


 


AGGGATGGGGAGGTGAAGGGAGGGAGATTGAATTCATAGAACTGCTGTGTCA


 


AAGGATGTATAAGTATATATAATGTATTGCCACCTCCAGAAAGGTATTTTATTT


 


ATTTATTTATTTATTTATTTTGTTTGTTTGTTTGTTTGTTTGGAGCAGAGAAAGG


 


TTTATTGCAGGGCCCAGCAATGAAAACGGGTGGCTTATGCTCAAAAACTTTGA


 


ACACCCTGACGGTACCTGGGAAAAGTTTTTATAGGTAGAATTTGGAGTGAGGG


 


CTGCAGAGGGTGTGCCCAGAAAGGTATTTTTTTAGCTTTATTATTTTTTCAAGC


 


TGTTAAGCCATTACTGTCGTGAAGTCAGAAATGGTTCCACGGAGTCTATAACTT


 


CTCTTGGTCCCCTTTCTGTGTTTGAGATGGCAAAGGCACATAAAGTCATGGAAA


 


AACTGAAAATGTATACTTCTCTTCTTTTAATAGGTGTCAATTGAAGTTGAAACT


 


CCTACCCAGATATCTTTAATTGATGAAGCATCAGGCGAGGTAACTAAGTACTA


 


AATACTGTGTTTTTAAAAAAATCATTACTTAGCATTAAATGATTGTGTACCTTT


 


CTCCTTGCTTGTGAATGCTCATTTAACCATAAATAAAATTTATAGTAAATGAGT


 


GGGTTTGTAGGGAAAAAAAAAACCATCTGTGATATATTTTCTATGTCCGAAGG


 


ATTAGTCTATGTCAGAGCTGCTTCTTTTTAGTATTACGTGCTAATTATAGGCGCT


 


CCGTAATCATGATATTACTAAAGAATTCTAATTCTAATTCTAATTCTAATTATA


 


ATTCTACTAAAGAATGTGATTGTCACATTTATTAAGTTTATTATAATGTTCAAG


 


ATGTAAAATGGTGCTCAGTGTTTTTGCAGCGTATTTAGGTATATTTTGATTTATT


 


TCCCCCCCCGAATCATTTTTTTATTCAAGAAGACATTATCCAGGTGGATCCATT


 


TTATTTTTTGTCCTTAAATTTAGAAGCAGGTCCTTTTGTGTGGCAGTTATCTTTT


 


TTAATAGTAAATAGGACTAAGGACCTACCTTTTATGTATTTATATATTTATAGC


 


TCAAATCATAGGTAGGGCTAGAAAGTGTTTTCCTCTTTTATGGCAAATCTAGTT


 


AAAATTTATTCTGTAGTCTGATTAGAACCTGCTTAATTGACACGTTTAATCTGG


 


AGGGTGTCCTTGGTGATGTGACTATAATGGGACAGGGGTCACTTTTTTTTTTTT


 


TTTGTCTTTTTGCTATTTCTTGGGCTGCTCCTGCGGCATATGGAGGTTCCCAAGC


 


TAGGGATCTAATCGGAGCTGTAGCTGCTGGCTTACGCCAGAGCCACAGCAACC


 


CAGGATCCGAGCCACGTCTGCAACCTACACCACAGCTCATGGCAACGCCGGAT


 


CCTTAACCCACTGAGCAAGGCCAGGGATCGAACCCGCACCTTCATGGTTCCTA


 


GTCGGATTCGTTAACCACTGCGCCACAACGGGAACTCCAGGGGGGTCACTTTT


 


TACATTTCCTCCCTGGAGATTAGGCATTGAAGTCCTTTTTTTTTTTTTTTTTTTTT


 


TTTTTTCTGTTTTTCTTTTGCTTTTTTTTCTTTTCTTTCTTGTCTTTTTTCTTTATCT


 


TTCCTTTCTTTTTTTTCAGTTTTTTATTTTGACTGCCTCACAGCACAGGGAGTTT


 


CTGGGCCAGGGATTAGATCTGAGCTGCAGTTTCAACCTTTGCACCATGGCAGG


 


GTCTCCTGAAGTCCTTCTAGAGCTACTGTCCCGAGGGAGGGGAGGCGGGATGC


 


CCTGAACCTTGCTAAGTGCCATCCTGAGCTCGGGTTGTGCACACGTTCGGGAG


 


ATGCTCTTGGTGACTGGCAGCCTGTGACATGTGCTGGTGAAAGGCTTTCTCAGA


 


ATTGGGCTCCAGACAGCAGGTGGCTCTTAAAGAGCAGGGTCCTGGTGCCCCAC


 


TCTCGTCCTTTCCTTTTCTTGTCCTGCACTGAGGCTTGCATGTGTGTGTCTCCAG


 


CTCCAACCTGGGGGAGAGCCCATTTTATATTTATTTTTATTTTATTTTAATTTTT


 


TTTTATGGCTAAGCGTGCAACATATGGAGATTGCCAGGCTAGTAATCAAATCT


 


GAGCCATGACTGAGACCTATGCCAAAGTTGCCCCAATGCTGGATCCTTTAACC


 


CAATGTACCAGCCTGGGGGTCAAACCCAGGCCTCCTTAGCGACCTGAGCCACT


 


GCAGTTAGATTCTTAACCCGTTGCACCACAGCAGGAACTTTTTATTTTTATTTT


 


ATTTTTTTAATAAATTTTATTGGAGTATAGTTGACTTAGTTGTATTTCAGGTATT


 


CAGCAAAGTACATCGGTTATACAAATACATACCTTTTTGGATTCTTTTCCCATA


 


TAGGTTATAGCAGAGTTTTGAGCATAGTTCCCTCTGCCACACTGTGGGTCCTTG


 


TTACGCAACTCTTTTATACGTAGTAATGTGTATTTGTCAGTCCCAGCCCCCTAA


 


TTTATCCCTCCCACCCCACATGGTAACCCTAAATTTGGTTTCAAAAGGGAGAGT


 


CTGGAGTTCCCATCGTGGCTCAGTGGTTAACGAATCCGACTGGGAACCATGAG


 


GATGTGGGTTCGATCCCTGGCCTTGCTCAGTGGGTTAAGGATCTGGTGTTGTGG


 


TGAGCTGTGGTGTAGGTTGCAGACATGGCTCAGATCCCGCATTGCTGTGGCTGT


 


GGTGTAGGCCGGCAGCTCCAGCTCCGATTAGACCCCTAGCCTGGGAACCTCCA


 


TATGCTGCGGGAGCAGCCTTAGAAATGGAAAAAAAAAAAAAAAAAAAAAAGG


 


AGAGTCTGTCTAAAGGGGCCTTTGGAATAAACGATGTCAGACACACCTTATGA


 


CTAGAGAGCTGGAGTGTCCAGAGCTCTGCCCCACCATGTATCAAACCTTCAGA


 


GGCTATGCGTGGTGCCCATGAGGATGTGCCTAGTGGGGCAGTACAGCATGCTG


 


TGTGGTGACTTGGGACCAGGCTACATCAGAGTTTTAAAAAATGTTTTAAAATA


 


ACTTTATTGAGACATCATCCACGTACCATCCCACTCACCCCATTCAAGTTCACA


 


GTTCAGGAGTTTCCATTGTGGTGCAGCGGAAACATACCCAACTAGTATCCATT


 


AGGATGCTAGTTTGATCCCTGGCCTCACTCCGTGGGTCAGGGATCCGGCATTGC


 


TCTGAGCTGTGGTGTAGTTCCCAGATGCACCTGGGGCCTGGTGTGCCTGTGGCT


 


GTGGTGTAGGCCAGCAGGTGCAGCTCCGATTTGATCCCTAGCCTGGGAACTTC


 


CATGTGCCGTGGGTGTGGCCCTAAAAAGTGAAAATAAAAATAAAAGAAATAG


 


GCAGTTCAGTGGCTTTTTGTATATTCACAGAGTTGTATATCCACCATCACAAGC


 


AATTTCAGAATATTTTCATCACCCAGAAGACCACTTGGTACCCATTAGCAGTCA


 


CCCTCTATTTCTCCCTCCCTCTGGCTTCTAGTAACTACTAGTCTAGTTTAAGGCT


 


CTATGGTTTGCCCATTCTGGACATTTCATAAAAATGGACTCATATAACACGTGG


 


TCTTTTGTGAGTGCTTTCAGTTTGCATAACTTATCCATGCTGTGGTGGTTGTCAG


 


TACTTCATGTCTTTTTATGGCCGAATAACATCCCACTGTATGGCTAGACCACCT


 


GTTGCGTATTCAGTCATCAGTTGATGGACATTCGGGCTGTTTCTACTCTTTGGC


 


TGGCATGAAGGATGCTGCTCTGAATATTCATATATAGGTTTTTGTGTGTGGACC


 


TGTTTTCGTTTCTCAAGTGCAAGAGTGAAATTGCACTCTTGCACTTGAGTTTCT


 


CACAGCAAGAGTGAAATTGCTGTGTCATGTTGCAACTCTGCATGTAACCATTTG


 


TTTTGTGGGTGAGGATTCCACTTGTTTCCAGAATCTGGAGAGTGGGGAAATGA


 


GGAGAAAAGGCATACGTCCTTGGGACGTACTTGCATCTGTTCTGTTAAGGAGG


 


GCTTTGTTTAACTGGGTCCTAATGATAAATTTCTATTATTTTTTTTTCTTTATTTT


 


CTTTTTAGAAGGAAGAAATTGTTGTGACTCTCTTACCAGCTGGTCATTGCCCAG


 


GATCAGTTATGTAAGGGGGTTCATCTCTTTCGCCCATTTATTCTGCGTAGAAAC


 


GTATTGTATTTGTAGAAATAAACATTGAGGGTCTAGAAAACAATATGTGTAGG


 


GAGATTTGATTCTCTTGATGGCAGTTCGTTTAATCTTTGTTTCCAAATTGAATTT


 


GGGGGACTGTGGTGCAGATCAAGTTTGAAACGTGGCTTTCTGACAATGCAAGA


 


CTTCAGTAGCCATTTCTCCACTGGCTTTGCATTTTCCTGTCGTGTGGATTTTTCT


 


CTCCACAAACAGGAATGATTTCATTGTACAGTTGAGGGAGCTCACGTATTTTGG


 


GGCCATCGCTTGCAAACTCAGTGGCATCTCCATCATGTTATTTTTTCCAACCTC


 


CCGGGAAGAGGTGGCAGCTGAGTGTCTGGTGTGGTTAAGCAGTACGGAAATTC


 


TGTTCATGCCATGTCAGGGTGGCTGTGTTTGGCAGGCAGCGTGGGCTGTGGCTT


 


TAATATATATTTGCTTTACTGGGATTTTGTATGAGACCTAAGTCACTGAGATAT


 


ATTTCGTTTTTCAGGTCTTATTTCAGGNGCACACATGGAACTGTCTTGTATACA


 


GGAGACTTCGATGGCAAAAAGGAGAAGCTGCCAGAATGGAGCTTCTGCACTCG


 


GGGGGCAGTACCGGGCTTTATATAATACTCGAATGTTAAGACTATGTTGTTGTA


 


AAGATTTTACTGCTCTTCCCCCCTACACATGTATGAGGCTTTGTTGCTTTTATTT


 


TTTATTTTTTTTGCTTTTTAGGGCCACACCCGCAGCATATTGAGGTTCTCAGGCT


 


AGGGGTCGAATCAGAGCTGCAGCTGCCAACGAACACCACAGCCACAGCAACG


 


CGGGATCCGAGCCCCACATGTGACCTACACCGCAGCTCACAGCAATGCCGGAT


 


CCTTAACCCATTGAGCGAGGCCAGGAATCGAACTCGTGTCCTCATGGTTCCTA


 


GTCAGATTTGTTTCCAGCTGTACCACAACGGAAACTCCTGCCTTTGTTTCTATT


 


AGAAGTCTAACTCAATTATAATATTTGCAACAGCAAAGCAAATTTTTCCTCTTG


 


AAATGAGGGGATGATGACATTCTGTGTGTGAATTGGGGTAGTAGTTACATGAA


 


CTGATCGAAATTCACTGAACCAGGAGTTCCCTGGTGACTCAGTGGGTTAGGGA


 


CCTGGCATTGTCGCTGCTGCAGTTTGGGTCGCTGCTGTGGTGGGGGTTCAGTCC


 


TTAGCCCAAGAATTTCCATATGCCCTGGGTGCAGCCAAAAAAGGTAAAAAAAA


 


AAAAAAAAATCCCTCACCCATCTGTCTTATATATTATCCTATGGAAATCACTTC


 


CTAACCATGTCGATACAGTAAAAAATTCCAGGGAGCTCTCTTGTGGCCCAGTG


 


GATTAAGGATCTGGCTGTTGTCACTGCAGCGGCATGGGAGTGGCCAAAAAAGA


 


AAACCCAAGTGGACTGAAACCTCCTGTAAAATAACTGAGAGTTTCGACTTCAC


 


GCGTGTTGTATTTCACGTGATTAACAGCTCTTGTGTTCTGTTTATCTTCAGTGTG


 


AAAGACATCCAGAGTGTGTACTTAGACACCACTTTCTGCCATCCAAAGTATTA


 


CCAAATTCCCAGTCGGGTACGTCTCTCTGGACGGGTGGCTGTATTTCTCAGGGG


 


GCGGGCCGCAGGCTAACAGGTCGGGTGGTAACGGGCCCCCTGGCCTTAATGTC


 


TGGGGACGCTGGGAACGGACAAGGCCTCAACTGCCTCTTCAAACCCCTGCCCA


 


GGAGGAGTGTCTGAGAGGGATCTTGGAGCTGGTCCGCAGCTGGATCACACGGA


 


GCCCCTACCACGTGGTGTGGCTGAACTGCAAAGCGGCCTATGGGTACGAGTAC


 








CTCCCACCCCCACCCCCAAAGTCCCCTCTCACTGGAAACCCTATCAGATGGCCC


 


AGCCTTCCTCACCTTGGTTTACTCACTTCCGCTTGGGAGGACTTGACAGGTGGA


 


AAGAGCCCTTCGGTGAAAAGTCCCACAAAGGAAGTGTGTTTTGTTCAAAATAC


 


TGGGTCATGAAATAAACGTAGTGTTTGTCACGAGCATTGAAAATAAATGTGAA


 


CTAGGAAGTATCCGAGAGTATGGCTTGCAGTCAGAGTGGTTATTGTGTGAATA


 


TTTACATGGGGAGTCAGATATATCAACAGATCTTTCTCGACTTAACCAAAGGG


 


CTGTGTCCCAATAAAGCCATAAATCTGTCTGGCTCGAGTTCCCTGTAGAGGCTC


 


AGTGGTTAACGAACCACACTAGCATCCATGAGGGCGCAGGTTCGATCCCTGGC


 


CTCGCTCAGTGGGTTAAGGCTCCGCAGCATTGCCATGAACTGTGATGTAGGTG


 


GCAGACGCGGCTCGTATCTGGTGTTGCTGTGGCTGTGGTGTAGGCGAGGGGAT


 


GCAGCTCTGATTGGACCCCTAGCCTGGGAACCAACATATGCCACAGGTGTGGC


 


CCTAAAAAGACATTAAAAACAAACAAAAAATCTGTCAGGCTCCTCCCCTGGCA


 


GAGGAAGAGCCTCCTCCACTCTCCCTCTCCCTCGGGTTCTCACTTGCACACAAC


 


AAGGATCTGGAGATGCAGGCTGGCAGGTCCTGTAGCCGAGCCGGGATAGAGC


 


CTGCGGGCTTGTCGTACAAGGATGGAGTCCGGTGGTCTGGCAGGAGGTTGTTC


 


CTGCCGGGAGGTGCTGCAGCAGCGCCAGGGTAGAGTCAGGGGTCTGGCGGCA


 


CAGGGATGGAGACAGGCCCTCTCTCCAGGCAAGAGCGATCGGCTCAGGACGG


 


GAGCGGTTCTGAGTGGAAGGGAATTTCCCTTCAAAGGGACACCACCTTCTGAC


 


CTGGGCTCTTTTAACGACCCTTTCAGATCCATGGCCTCAGAGATCACAGTCTTG


 


ATCACAGTTGAAATCAGGTCACCATGAACCAGTTTTATGGACGCCTTCAGAGG


 


GTGGGCCACCCTGGGCTGGGGAATGGTCCTGGCTGCCACTGGGTGTGGGTGTT


 


TCCTTAGGTCCCTACATCATGGACCTGTTTGGGGACCCACATTCTCTTGCCCCA


 


GGCCCACTAATTTCCTGTCCCAGCCCCCGCCCCCGCCCCCGTGCTGTGTGTAAG


 


GGCTGAGTGTTGTTTTGCTCTCTGCATGGGGGTGGGTTTGGATCCTCTGGTCGG


 


TCCCACCTTAGTCACGTGGGGAAAGGTTATGGGGAGGCGCTCCAAGCGCACGC


 


CCCTGGTGCCTGCGTGTGTGTTCATTGTTTACGCAGGCCTCCTGTTTTTGGCAA


 


CAGCAAATGGCCTGTTTTGAATCACCCAGGTCCCCGTCCCTGTCCTCTCCTAAC


 


AGAGCCATTGCAAATAGCACGTATAAGTTAAACATGCATTTAAATAAGTTCCT


 


TTGTATCATTTTTAAAAATTAAATCCCACACGCAAAGGGACTTATTTAAAAAAC


 


ATTCTGAGATTGTTGTTGCTGTGTTTTTTGGTGTTAAAATGTCTTTTCAATTAAA


 


CCACAGCGATAATAGACTGTGTGTGTGCGCATGTATACACACGCACACGTACA


 


CACATCTTTATATATGCTCTGCTGGGTGCTGGGTTTTTTTGTTTTTATTTCATGG


 


CTTCTTGACAAAATATAGAGGTACCAGCCACTTACCCGTCCCAAATTTGCTGTG


 


TAAATTTTCTCGATGTGCGAAATGGAAAAGACCCGGAAGCTCCCGCTCTGAGA


 


CCTGGGGGAGAAACCCCGGGCTGAGGAGGGGGTGGGGAGGGAGGCAGGATTT


 


CAGTGTGTGGCTCCCTCAAGTCTCTTAATCCCGCTGTAGGGATACAATGCTTGT


 


AGACAACAGTGAGGTTTTTTTGGACAAAGAGACCTGAGAGGTGATTTATGACA


 


GCCTTTTCAAGCATCTTCATTTTCACGAGGATGAGCGTTTGTGTCTCTCTGGTG


 


CATCTCCGAGTTTACCTCTTTTTCTCTCCTTCCTAGGCCGGAAAAGTCAATTCAT


 


TGTTCCTGTTAGTCGTTAGTAAACAGGTAGTGAGAGGACTAGGCTACTGGATA


 


AGTAAGCAGTCATGCCTGGCTGCTGTGTGACAACAGTCCTGGTAGTAATTACC


 


CCAGCAGTTAACATCCTCCTGGAGTTCCCGTTGTGGCTCAGTGGTTAACGATTC


 


CGACTAGGAACCTTGAGGTTGCGGGTTTGATCCCTGGCCTCGTTCAGTGGGTTG


 


AGGATCCAGCGTTGCCGTGAGCTGTGGTGTAGGTCGAAGACGTGGCTCGGATC


 


CCGCGTTGCTGTGGCTCTGGCGTAGGCCGGCGGCTACAGCTCCGATTCGACCC


 


CTAGCCTGGGAACCTCCATATGCTGAGGGTGCCACCGTAGAAAAGACAAAAA


 


GATAAAACAAAACAAAAAACTTCCTAATCTGTTTTGTTGCTCAGACTTCAAAAT


 


GACAATACTGTTTCTTTTTTTTTTCTTTTTCTTTTTCTTTTTTTTGGTGCCATTTCA


 


TTATCTCTGTGTTCATTTAGGTACTTGTTGTTTATTCTGAAATACCCAGAAAAG


 


ATTTAAGCAAAACTGACATGTTGGTTTTAAGTTTTAAGCAGTATTAGCTTTAGG


 


AGTTCCCTGGTGGCCCAGTAGGTTAAGGAACCAGCATTGTCACTGGAATGGCT


 


TTGGTTGCTGCTGTGGCACAGGTTCGATCCCTGGCTGGGGAACTTCTGCATGCC


 


ACAGGTGTGGCCAAAAAAAACTCCAAACAAACAAAAACAAAAATTAAAAATG


 


AAATCATTAGCTTTAAGATTCTAATGGTGGCTGAGATCTTTTTTTTTGGTCTTTT


 


TTCGTCTTTTTAGGGCTACCACACCTGCGGCATGCGGAGGTTCCCAGGCTAGGG


 


GTTTAATCAGAGCTGTAGCCGCAGGCCTACACCGCAGCCACAGCAATGCCAGA


 


TCCGAGCCGCATCTGCGACTGACACCACAGCTCACAGTAATACTGGATCCTTA


 


ACCCGCTGAGCGAGGCCAGGGATCAAACCCGCAACCTCATGGTTACTAGTCTG


 


ATTCGTTTCCGCTGCACCAAGACAGGAACTCCTGAGATTTTTTTGTTGTGTTTTT


 


CTGCATCTGCCCCCTCCCCCTCTGGCCAATCCACTGTGGCATAGGAGGATCCAG


 


ATCTCGGCTTCCTTGTTACGCTGGCTGCCCACCACCAGCTGCCCTTAAGTAGCA


 


TGTTGTATGTGTCTTCGAGAGCAACCTTCTTGATTATTTTTGTCTGCTGTATTTA


 


CTGCAATACTCCCAATATTTAGAATAAAGCTTTTTTTATTGATGATTTTTATTTT


 


TATTTTTCTAGCACAGCTGGTTTACAGTGTTCTGTCCATTTTCTACTGCACAGCA


 


AGGTGACCCAGTCGTACATACATATATACCTTCTTTTTTCTCACATCATCACGC


 


TCCATGATAAGTGACTAGATAGAGTTCCCAGTGCTACACAGCAGGATAGAATA


 


AAGCTTGATGTAGAGCAGGTGCACAGTAAATTTTGGTCGAGTGCCTCTTGCTA


 


AACTGAGGGACGGATTTACCTGATCCAGTGAAAGCCACCTACAGGAGGCGCTA


 


AATGCCGGGACCATGATCTCAGCGTCCTGGTGCAGTACTAATGTGCGTGGAAA


 


GAGAGGAAAACTAGGTCTCACGGCAGAAGAGAGAGTTCATTCTAACTGGATG


 


ATTTGCCCCGGCCTAGTTACCCTTGAAACACTTTGTTTACAGGAAATACAATTC


 


CAGTCATGTGCTTATTTGTGTTGTTTTCCTCAGGTTCACGTGAATAAACTGGAC


 


ATGTTTCGAAACATGCCTGACATCCTTCATCATCTCACAACAGACCGTGGCACT


 


CAGATCCATGCCTGTCGGCATCCAAAGGTACACAGTAAACTGCTCTGTTTGCG


 


AGATCCATGCAAGAATTTCAGGTCCAGGAGGAACCGGCGAAATTCCATTTTCT


 


AGGCCCTTCAGGTCGCAAATGTGAAAACTGAGGCTTAAGGTGTTAACCCAAGG


 


TCATTACTGTATTGCAGTACCCTGAACTTCATAATGAGTTAACAGACACAGAC


 


ACGCCATAGTTTTGTTTTGTTTTGTTTTGCTTTTTAGGGCTGCACTAGTGGCATG


 


TGGAAGTTCCCAGGCTCTTGAATCGGAGCTGCAGCTGCTGGGCCCACACCACA


 


GCCACAGCAACGCCAGATCTGAGCCTCATCTGAGACCTACACCGTAGGTCACA


 


GCAACGCCGGATCCTTTACCCACTGAGCGAGGCCAGGGATCAAACCCACATCC


 


TCAAGTGGCGGATACTAGTTGGTTTAGTTCCCGCTGAGCCACAATGGGAACTC


 


TGGTTTTCAACCTTTTAAAAATACTTGTCACTTTTTCTCCAACTTTATTTTGAAA


 


AATTTTAAAGCAGTATAGTAGTTAGACTAGTACCAAGACCTCTGTGTACTCTAT


 


TTTTTCTAACTGGCGTATAGTTGCTATGTACTGTTACGTAATTTACAGGTGTAC


 


AGTGTAGTGATTCACAATTTTTAAAGGTTGTAATCCATTTATAGTTACTATAAA


 


ATATTAGCTATATTCCCCCTATTGTATGATATACTCTTGTAGCTTCTTTATTTTT


 


TAGGTAGTAGTTTGTACCCCTCAACCCCCCACCCTGTCTTGCCCCTCCCCACTT


 


CCCTCTCCCCACTGGTAACCACTGATTTGTTCTCTATAGCTGTGAGTCTGCTTCT


 


TTTTTTTTGTTATATTCACTAATTTGCTATATTTTTTAGATTCCACATGTAAGTG


 


ATATCACACAGTATTTGTCTTTCTCTGACTTATTTCATTGAGTGTAATGTTCTCC


 


AAGTCCATCCGTGTTGCTGCAAATGGCATTATTTCATTCTTTTTATGGCTGACA


 


TTCCATTGTGTGTGTGTGTGTTCCACATCTTCTTTGTCCATTTATCTACTGATTG


 


ACACTTAGGTTGCTTCCATGTCTTGGCTCTTGTAAACAGTGCTGCTGTGGACAT


 


GGGGATGCAGTATCTCTTTTAATTAGTGTTTTTGGTTTTTGTGGGTATATACCCA


 


GGAGTGGAATTGCTGGCTCCTATGAACACCGTGTCCTCTTCTTCTGGATTCCGC


 


AGCTGCCCAGGTGTTAACAGTTTGCTTTCTGGCACCCTCTGTGTCTGTCTGTCT


 


GTCTCTCTGTGTAAGCAGCACCCCACCGTGCAGCCCACTTGAGAGTCAGATGC


 


AAACGTCATGACACGTCACCCTAAACAAAGTCGTATAACCACAGTACACTTCC


 


CACACTCAGGGAATTTAACATGAATGTGATACGACTGGGTATGATATGGTCCA


 


TATTCAGATTTCCTCAGTTGTCTCAATCCCTTATGGCTTGGTGGAGTGGGGAGC


 


AGGGCCATCCAGAATTTAATTTAAAAACATGTGTTGCATTCACTCAGGCTGCTT


 


TAGTCTTTTGATGTAGATCAAGTCTCCAGCTAAAGTCCAGGCCAGTTGTTTTCT


 


AGAATATTCCTCAATTCGAACTTACCTATTTCTTCATGTCTATATATGTATATAT


 


ATATTTTTTTTTTCATGCTAAAAAGTCATCTGCATTTTTTTGAGACCCTGGTACA


 


AATATATTTGTTGAAAATGTTAAACCATTTTTCTTATTTTAAAATCTTTTTTAGG


 








AGAATTCCACTCCACATAATCAGTATTAAGCCCTCCACTATGTGGTTTGGAGAA


 


AGAACTAGAAAAACCAATGTTATTGTGAGGTAAGCAAGCAGCGTCTTTTGAGA


 


GGAACCTTGCTTTGAGGTAAATCGATAGTTTAAAGGCAGTCTAGCCTAACCTC


 


AAGAGGGGGGCATATCATGATTGTGGAAAATAACTTTTGAAAGTTAAACTCTG


 


TTTAAATTAAAGAGAAGCGGCTTCCAAAAGCTATCAATTGCTTACTACCATGA


 


GCCAGTATTTCCTGGGGTCTACTGAGTTTGACTGAGAAGATGGTTAGAGGCAG


 


GCTGTCTTGCTTGGCCGGAAGTTGGGAATTGACCTTGAAGGGGTTGGAAGCCC


 


TTAGTGGAGCAGAGGAGGCCCATACTGATCCTCATGTTTTAGCTCATGTCCTGA


 


AACATTGGCCGTTTGGAATGTTCACAGATATTTACATTTATGGAAAGAGTTCCC


 


TCCTGGCCCAGGAAAAAAATCCTTGATGGCAGGGTGATTTCATCCTGCAGTAG


 


CCTAAAATGAGAAGACTCGGAGTTTGGAACTTAGTTTTGATGAGTGACCCTTA


 


ATTTTGGGTTTTCCTTTCCCTTTAGAACTGGAGAGAGTTCGTACAGAGCCTGCT


 


TTTCTTTTCACTCCTCCTACAGTGAGGTAAGAGGATCCCATACTCAGAACCTCG


 


GCTGCTGAGGCATCTCTCTTCCTCCTGCTTAAGACTGGTGGGGACTCTTGCTCA


 


GGAAACACAGGCGAAGACATCTCGGTGGATGAGCTACGTTGCTTAAGTAGAAA


 


GGAAATGATCACCCCTTGTATCTAGCCTAAACTTTGAAGAAAATGTATGAATA


 


AGGTTTTATTCCCAACCTGCTTTCTAAATGTATTTTAAACCCCTCACTTCAGTTC


 


TGTTCACCCGTCGTAAAAAAATTGTCAGTATTACTATACAGCAGATGGAAATG


 


TGGTCAGTAGCTTGTGATTACCCATACTGGAAAAGAATCTGAAGCTCTATGCC


 


CGAAACTCACACAGTATTGTAAATCAACTATAGTTTAATTAAAAACCCCAAAC


 


AAAACCCCAAAAACTTCTTCAAGAAAGTAATAGTTAAGAATATAAATGAAAAG


 


ACTCCCAGAGTTGGTGCAATGGATTAAGGATCCAGCATTGTCTCTGCAGTGGC


 


TTGGGACGCTGCTGAGGTGCCAGTTCGATCCCCGGCCCAGGAATTTCCACATG


 


CCACAGGTCCAGCCAAAAATTAAAAAAGCATTTAAGACTTAGAGACTTGAAAT


 


AATTTGACACAAGCCCACCAAGATCTAAAAATGACAAAGCTGAAGCTCGTCTG


 


GGTTATAGAGATGTTTTGAGGAAGAGTGACAGAGTTCCTTTGTTTAACATGCTG


 


AGCTTTGCAGAAAATCAAGGTATTCGTGAAATTTTTGCATGTAGTTGTCCTTGT


 


TTGGATTAGAATAAACCATGAAGATGAATCGAGCAGTCTCAAAGATGTACTTC


 


CCCTTGGTATTGCTAACAATTATATGTGGGAAGAAAGAAATGCAATTCTAAGT


 


TCTTTCCCTGTGCATATTAAATACTTGGCTAAATTCTAACTTCCTTAAAGGGAA


 


TTTGTTAAATATAGGGTGAGTGACTTTAGATAAGGAAACTAGGAGTTCCCGTC


 


GTGGTTCAGTGGTTAACGAATCCGACTAGGAACCATGAGGTTGTGGGTTCAGT


 


CCCTGCCCTTGCTCAGTGGGTTATCGATCCGGCGTTGCCATGAGCTGTGGTGTA


 


GGTTGCAGTCACGGCTCGGATCTGGCATAGCTGTGGCTCTGGCTCTCAGCTTTG


 


GCTCCCCTAGACTGGGAACCTCCATATGCCTTGGATGTGGACCTAGAAAAAGA


 


CAAAATAAAAATAAAAAATTAAAAAAATAAAAATAGATAAGGAAACTACTGT


 


CCTATAAAGAAATAGAGAATCTTTGAATCCCATTAACAAGTGAGACTCATTCC


 


TTACTGGGGAACAGAATCACCTGAAGAACATTTAAATATATTGATTCCAGGTC


 


CCGCTGTAGCTCAGTGTTAGAGGCTCTTTGGGGCTGGGGCATGTGTGTTGGGTA


 


TTTCTTTTTGAGAGCCCCGCTGGGTGCTCCCGTGCACCTGGCATTAGGAACCAC


 


AGCCTTGAGCGACCTCTGCTCTCTTTCCCCTGGTATCCCTTTATCTGTTGCTCTT


 


ACACCCAGTTTTTTCACCATAGAGATAACTGATGCATTTTATTTTCCTTCTTCCA


 


GATTAAAGATTTCTTGAGCTACATTAGTCCTGTGAATGTATATCCAAATGTCAT


 


TCCACTGGGCACAACTCTGGAGAAAGTTAAAGAAATGTGAGTCATTAGTACTT


 


GCGGAACTTCTGTGGTCCAATGGGATGGATCCAGAGGATAATTTCAGGCCTGA


 


AAATGGGGACAAGGCTGTAAAATGGACGTGGCTGTCAGTAGGGTTCTGGTTGG


 


GAGCATCTGGGCTTTTTCAAGTGAGTGCGTTTTATATAATCTTAAAAGCTTCTG


 


GGCATCTAGCATAATGGACTGGTTGGCAATGAAGACCTTGAAAAGGGCGGGAC


 


ACCTTCAAATCTTATTTCTGTGCCCTTTTGTATTTTATTTTCACTATATCTTAGCT


 


TTCAACCTCTAGAATGAAGAGATCAAATTTGTGAGATTCTTGCAAGAGATAAC


 


CCCATTAAATAGCGAACAGGAGGCAGTTTGCTGTACTGAATTCCCTGAATGCC


 


AGAAGGTGTTACCCTATTATTTAACAGTTCATCAGAATAGGGACATGCTTTTCT


 


GCAGTGCAAGAGCAAAGATAAAAGTTTTGCTTTCCGTCAGTCCTACAATTAGA


 


TGGGTTGTTTTAAATGCTTATATTTACTACTTTATTATATTGAAGTAATTTTAAA


 


TTATTAAACATTGCTGAAAAGTTTGTTTTTTTTTTTTTTTTTTTTCTTTTTTGGCC


 


TCTCTGTGGCAGATGAAGCTCCTGGGTCAGGGATCAGATCTAGCTGTTGTTGTG


 


ACTTAAGCCACAGTTGCGGCAATGCTGGATCCTTAACCCTCTGTGCCAGGGTG


 


GGGATTGAACCCACGTCCCAGTGCTCCCAAGACGCCACTGATCCCATTGCGCC


 


ACAGAGGGAACTCCAAACATTGCTGAAAAGTTTTTAAAAGCAGTGGTATGATT


 


TCCTCCATAGTCAAATTTTTATAATCTCTATAAATGATGCTTCTATTCCCAGCTT


 


TCCCAACAGGAAAAATAATATCTGTATTCATTTCAGCTTAAAGCCTTTATGCCG


 


ATCTTCGCAAAATATCGAGCCAAAGTATAAACCACTTGGAAAATTGAAGAGAG


 


CTAGAATAATCCATCTAGACTCAGGTAAGATGAATGACCCTGGGGTCAGAGGT


 


GTGGGTTTCTCTGCAGGAGCACTTTGCAGAGTTCTCTGGCCTGGTGAAAGCTGC


 


CCTGGGCAGAGTTGGACCCAGGATCCACTGTCGCTCAGACTTGTCATGTGGTCT


 


TGTAACGTGCTCTGCCCCCCTACCTTCAACCTATAAATGTAGGAGTCAGATTCC


 


CTTCACAGGTTTAGAAAAAGTGATAAAGAGAACTTAGTCTGTATTGAATGTTG


 


TCTTCAGCTTTCTGGGAATGTGCCCTGCAGCTTGTATTGGCCATAAGGAGAGGC


 


TCTGCTGTTACCCTTGAACATTTCCTTCAAGATACTAGTCAGTGCAGATAATGA


 


GAACTTTGTCTCCCATCATTTCCCATCACATTGATGTGTAGTGTTTATACGAAT


 


GGATTCTCTATACCTTCCTGCCTTCTTTCATTTTCTGATTGATTTTTTAAAATGA


 


AGTATATTTGATTTACAATGTTGTGTTAATTTCTGCTGTACAGCAGAGTGGTGT


 


AGATATATATATATTCTTTTTAAAAGTATTCTTTTCTGTTATGGCTTGTCCTAGG


 


ATATTGAATATAGTTCCCTGTGCTGTGGAGTAAGATCTTGTAGTTGATCCATCC


 


TCTCTCTATATATATAATAGCTAACATCTGATAACCCCAGCCTCCCACTCCATC


 


CCTCTCTCAATGCCCTCCCCACTGGTAACCATAAATCTTCTCTATATCCGTGAA


 


TCTGTTCACCTATTTGGCTTGTTTGCATTTTTGGTTATTACATATAAAGCTGCTT


 


TCATCTGCTTACTTTGGGTTTATTTTCCTCTTCATTTAAAATTTCTTAATGTGGG


 


AGATGAGGTCATTGATTTGAGCCCTTTCGCCCTTAATACAAGCATCTAGTGCTA


 


TAAATTTCCCTCTTTGACTTGTGTTGTCATTTTCCTCCAGTTCAAAATAGTTTCT


 


AATTTTTCTTTTCCTCCCTCCCTCTCTCCCTTCTTTCTTTTCTTCATTTTTGGCTG


 


CCCTGAGGCATATGGAATTCCCAGGCCAGGGGTCAGATCCAAGCTGCAGTTGT


 


GACTTATGCTGCAGGTGCAGCAATGCCAGTGTGCTGGGCCAGGGATCGAACCT


 


GCGTCCCAGAGCTCCAGAGATGCCACTATTCCCTTTGCGCCACAGCAGGAACT


 


CCTCTAATTTTTCTTCCTTGAGGCGCGGGTTTATTTACAGGTACATTATTTCGTT


 


TCTGAATTCTGGAGGAATTTACAAAGATAATTGTGTCATATAATTTTATTGTGG


 


TCAGAAAAAATACAGTGCATGGATTTTACTGAGATTCATGGTCCAGAATAATG


 


GTCCATCTTGGTAAATGTTTCATGTGTACATGAAGAAAATATGTATACTGCTGT


 


TGGGTGGAGTGTTATATAAATGTCTAGTTAATAATGTTCAAGTCTTCTGCATCT


 


TTGCTGATTAACTTGTTCTACTTGCTCTATCAATTATTGAACGTTTTTGAAATGG


 


TGAATTTGTCAAAATTTGTTCTTCCACTTTTTGTGTCATCGATTTGAATAATCTT


 


TAAAACTCCTATGTCCTCTAGAGGAACTGACCCTTTCACCTTTGGGAAATAACT


 


CCCTTTATCCTTCTTTTTATTAGCTTTGAAATCTACCTCACTGGATAATAATACA


 


TCCATTCCATTTTCTTCTGATGTGTGTTGGCATAGTATATCTTTTTCCATCTCTTT


 


ATTTCTCACCTAATTGTCTTTATATTTAAAGTGGGTTTCCTGTAGGCAGTGTATT


 


GATGGATCTTGTTTCTTTTTACATCCATCTGATAAATTCTGCTTTTCAGTTGGAG


 


TGTTTTCAGGCTACTTAGGTTAATGTGATTACTGACCTTGTTAGGTTTACCATCT


 


TGCTATTTGTTCCATCTGTTCTTTCTTCTTTCTCTTCTACCTTCTTTTGGATTGAG


 


AACGGATAGAGATTCCATTTAATGTCTTATTTGTTTTATTAGCTGTAACTCTTTT


 


ATTCTTTTAGTGGTTTACAGTTTTCATCTGTAACCTATCAGAGTGAAAATTTCA


 


AGATATATATTCTACTTGAAGTATGGTATAGGAAGGTGACAACAGTATATTCC


 


ATTTCTCCCCTCCCAGTCTTTGTGCTATTATTGTCATACACTTTATTTCTCTGTTA


 


TAAACTCCACAATACATTACTGTTTTTTTTTTTTTTTTAACAAAGCTTTAAAAAT


 


TGATCGCTTATAGGATAATTTGGGAGTTCCTTTTGTGGCTCAGCAGTAACGAAT


 


CCATGAGGATTCAGGTTCGATTCCTCGCCTTGCCAGTGCGTTAGGAGTTGGCAT


 


TGCTGTGAGCTGCAGTGTATGTCGCAGTTGTGGCTCGGATCCTACACTGCTGTG


 


ACTGTGGTGTAGTCTGGCACCTGCAGCTCCAATTCGACCCCTAGCTTGGGAACT


 


TCCATATGCCCTGTGGGCCTGAAAAGCAAACAAACAAACAAAAAACCAACTAT


 


AATTTATATGCCATAAATTTCATCCTTTTAAGGTGAACAATTCCATGATTGTTA


 


CTGTAACGAAGTAGGTTACGTGCTAAGGCCTCTTCTCTTGGCTTGTGGATGGGT


 


GCCATGCTGATGTGTGCTCACATGGCCTTTCCTGGGTGCTTGTGCCTGGTTGGG


 


AGGGGTGAGTAAAGGAGGGGCTGGGGGATATTTGCTCTCTAATGTCAGTTCCT


 


TCTTTGGAGGCCCCATTTCCACTTTGGGGATTAGGACTTCGACAGGAATTTGTG


 


TGGGAAGGAACACACACTCAGTCTAAAACATACAATAAAATTTACGCTGTAAC


 


ATGGTAAGAAACTGACAACTGTTCTTCAGACAGAACACATTTTCATTCTCACCA


 


GCAATATATACGGATTCCAATCTCTCCACATCCTAGCTCACACTTTTTAGTGTC


 


TTTGATTTGTAGTTATTTAATGATGTCAAATAATATATCTCATTGTAGTTTCACT


 


TCCTTTCTCTATTGATGTTGATCTTTTTCCATGTTCTTATTGGGCATTTATTTATC


 


TTTTCTGGTGAAATGTCTATTCAGACCTTTTTGCCTGTTTTTAAATTGGCAAACT


 


CATTCCTGAGTTGTTAAGAGTTCTCTCTATAGGCTGGGTATGTCTAGTATTATTT


 


GCAAATATCTTTTTCCAGTCTATAGCTTGTCTTTTCACTCTCTTCATGGTGTCTT


 


TTGAAGTGTAAAATTTTAAATTCATCTAATTTCTTTTATAGCTTGTGCTTTTGGT


 


GTCATATCGAAGAAGCTATTGCTTAATCCAAGATCATAAAGATTTATTTTCATA


 


CTTTATCATTTCTGGGTCAGTTTTGACTTTTCCCCTTATTTATAGGCCATTTTCC


 


CACCTTTCTACACATTTGGTAATTTTTTATTGGATGCCACGCTTCTTGAACTTTA


 


TGTTGTTAGGCATAGCATACCTTTGGATTTCTGAAAACATTCCTAAACTTTATT


 


CTGGCACGTGGTTAAGTGACTTGGAAAGAGTTTGGTTGTGTCTGGCTTCTGTGC


 


AAGCCTCTTAGGCAAAAGCAGGGCAGCATTTAGTTTAGAATCCACTTTCCCCA


 


CTGCTGAGGCTAGGCGCTTCTTCACACTCGTAGGAATAGCCCGAGTTATGAGG


 


CGTTACTGGCTCTTGGGGGCAGGTACTGTTTCCGTCACCGTGTGCATCCTCAGC


 


ACGCTTTCCTTTTATTCCTCGGAGTGATTCTCTCCCTGGCCTCACAGCCAGGCG


 


CTGACTATTCGAGGGGCCCCTTGTGCAGCTCATCAGAGCTCCTCAGTACTGCTC


 


TCTCCTCTCTGGCCTCAGCTCTGTAAACTCCAGCCAACCTGGCTTCCTTGGACT


 


CCCAGTTATATCTCAACTCAGGGATGCCAGTGGGCTCTCCTTGCCAGGAAACT


 


GTCACAAACCCATACGCTGAGGCAATCTTAGGTATCACTTGTCTTTGTCTGATG


 


TTTAATTCCTTGAGAGACTTTGTTTCTCATATTTTATCTGGTTTTTAGTTGACAT


 


GGGAGGTTAAATTTAGTCCCTGTTAACTCTTTCTTGGCTGGAAGCATAAGTTCT


 


TTTTTTTTTTTTTTAATACTTAAAATTTTGTACCTTTTATCTATCCAAATGATCCT


 


CGTTTTTAATGACGGTTTTATTTTTTTGGTTGGGCCACTAGTTTAAGAGTTCTTG


 


AGGTTATGTTCATTTCCACTTCTTCCTGCTTTTTCTTTATTAGCTTCAATTTTGAT


 


CCTTCTAGATTCCTTTGGTTCCTTATTCCTTATCATAGCAACTTTCATACCTTTT


 


ATTTGTACAGGTGTAGATCTTTCAACGACTCTTCAGTGTCCTTCTGTATTTTGTA


 


AGTCACAGATATTATCAGGTTTACTTTAGTTGCATGTATGGTACTGACTTTAAA


 


ATTCCTACCCTGTCCCATGCATGTACTTTAAGGTCTCCATGATGATCTCTGCCA


 


CTACGTTGACCAGTCTTTCACCTGTTCTAGTTCTTCTTTTTTAATGACCACATCG


 


GTGGCATATGGAAGTTTCCCGGCCAGAGACTGAATCTGAGCTACAAGCTATGC


 


TGTTGCTTGAGGCAGTGTTGCATCTTTTAACCCACTGTGCCGTGCTGGGGATCG


 


AACCTGAGTCTCCACAGTGACCTGACCACTGCATTTGGAACCTTTTCTAGTTCT


 


TAACATGCTCTGTTGATGCAAATTAGTGGTTACTATTACACATGCAGTGTTAAA


 


TCAGTTGCTCTGTGTTATTCTTTTCTCAGAATCTGAAGGGTACATGTAGAAGTC


 


GCTTGTCTCTTAGCATCTTTAAAAATATTTTAATTAGGAGTTCCCGTCGTGGCG


 


CAGTAGTTAACGAATCCGACTAGGAACCATGAGGTTGCAGGTTTGATCCCTGG


 


CCTTGCTCAGTGGGTTAAGGATCCAGGTTGCCGTGAGCTGTGGTGTAGGTTGCA


 


GACGCGGCTCAGATCCTGTGTTGCTGGGGCTCTGGCGTAGGCCGGTGGCTACA


 


GCTCCAATTAGACCCCTAGCCTGGGAACCTCCATATGCCATGGGAGCGGCCCT


 


AGAAAAGGCAAAAAGACCAAAAATATATATATATATATTTTAATAAAACTGTG


 


CTCTTTATATAAGTTTAATCTTTATCTAATTTATATAATTTAATAGTTAACCATT


 


ATTATAATTATCTCAAGACCAATTTTATAAAATTACTGTTCCTTTACTGTAGAT


 


AATTTAGAAAATAGTTATGTAGAACCTTACAACTATATCTTTATTAGCATTTTC


 


ATGTATTTCCTTTCCATCCTGAATCATTTATATAAATACCTACACAAATCACGG


 


GGGTGATATTGAAAATATTCAACCTTGGTCTCATTGGTACCTACAATGAGACCA


 


GCAGTAGCCGTAGTGCAAGACCATAGTGCAAGACCAATTTCTTCATTTGTGCA


 


GCTCTTAGTTCCTGGACTGTGAAGAGGTGGGCAAGGGTCCTTAGGTGTAGGGT


 


CCAGGGCAGTGGGGGACAGGAAGGACATCTGGAGCTGAAGGGAAGAGCCGTT


 


ACTAATTAGCCTGGAAGTGTTTACATAACAACTGTAAGACTGAATAGGTGTAT


 


ACAGACTGAAAGGCAGCTTGGATCACTTTGTGTGTATGTGTGTGTATATATACA


 


CATATACATATACTTAACCTCTTTACTGGAAAATTTTTATTCTTCAAAAGCACC


 


CGTTCAAGGGCTGCATATTTTAAATTGCATTATAACTATATAATTTTTGTCATGT


 


ACTTTGTCATATAGATTATGAACTTCAGTTTTATAAACATTGTGGTAAACATTG


 


TACAGAAGTCTGTGTCATAGCTCTCATTACTGGGTTCGATTATAGAAATTATGG


 


TGCTTGATATATACATATTGCCAAACTGTTTCCCAAAATGGTTATACCAGTTTA


 


TATAGACATCAGCAGTATATGATGGTTGAAAGTATCTATGAAGTATGACATTT


 


GTCCTTTTTAATTGAGTTTGGGTATGTTATCAGTTTGTGTAAATGTTAAGGATTT


 


CAACACTTCTGTAAGTACTGTGATTTTTCTGTGTCTATTTTCATAGAGAAATTTA


 


GTTTTTATATTCTAAAAAGAATACATGTCAGAATAAACTTCTAATAGTATAAAA


 


ACTTTGTTTTTCAGATACATAGAGGTGAGGTAATGTGAAAATGTTTTATTTTTA


 


CAGTTGAAACATACTGTAATGTTGGTATAGTCACTGATGCGAAACAGGAAGCT


 


GTCCACCTTAGCGATGATACAAAAACATTCCATTAAAAGATAATTTTGGCACA


 


TCAGTGGAGGAGCTATAACAAATGTATTAATCCATGGCTTAAAATGAAGCCAT


 


AACTTGGAAACTAGGATGATTCCAAATATAGTGGCTAGACATCTTAGTCACCTT


 


AAGTCCTTTAGAAAAGACGTAAGATGAAATAATTGCTTGTTGAAACAATTATA


 


AATTGTAAGAGAACACTAAGAACTGGCTCTCATCCTTAGCTCCCCCTGTCTGGT


 


TGCAGACCTTCTCTGGGCCTGTCTCCTTGGTAAAATGGGAGCTAGATGGTGGTT


 


TCTACGGACTCCTTTCTTTAAGAGTCCTGAAAAATGAAGTTTTTAGCTGTGTCT


 


TTGATTGCTGGGAAAAGTTAACTTATAATTTACTTGCTTTTAGCACTAGAACAG


 


TAAGTTGCCCTACACAGTAATCCTGTAAGCATTTAACTTCCTTCTCTGGGGGTA


 


AGTGGTATTAGAGGGCAGGAAAATGTTTGTTTTCCCCCAATGCCACGACATGT


 


CATTTTAGCTTATGTAAATTTCATGTATTGAATGATAAGAATTTCATTCTTGAA


 


GATTTCCTCATTTANAGGACACAGGTGGATATAAACATGCTCATGCAGCATGC


 


TTAACACAGTAATATGACACTAAAATGTGGCTTGTCTAACATTTTGGTTCAAGT


 


TGATATAATCTTTCTGTGTTTCCCCCAGTCTCATGGCCTTGTGCACTTAGCATAG


 


CCTGCGTAACAATTACCAGAGACTGGGCAACAACTTCTCTCAGTTCTGGATAC


 


CACTGGGATGTCCAGAATCAAAGTGCTGGCAGATGTGGTTCCCAGTAGGGGCC


 


ATTTCAGCCACCTTCTCACCTTCAGTGCACTCGCATGGCCTTTCCTTTGTGCATG


 


GGCATGGAAAAGTGAGAAATCTGCTTCCTTTTCTCATAAGAACACTAACCCCG


 


TCGCATAGGTGCCCCCCCATGAGTTAATTATCTTCTAAAGGTCTTACCTCCAAA


 


TACCATCACGTTGCACTTAGGGCTTCAAATTATGAATTTTGGCAAGACGTGGAC


 


ATTCAGCCTATAACATGCACTAAGTGGGTATCAAAAAATGTTCATGAGGGCAA


 


ACTTTAAAAAACAATTCTTTTTATTGTATCAGCCAACTTGCTGAGCATCTACTA


 


TCTGCAAAGCATACACAGTCTGCAAACCTTGCTTCTAGTCTAGGTGGTTTGAAA


 


ACCTGGCTGACGAACAGAATCAGCTGTAGAACTTATAAAATAAGGCAAAATCA


 


AATTATTGGATCCTACTTTGAGAGACTGAGTAGGTCTGAAACAAAGCATAGAA


 


AGCTGTTTTAGTTTAAAAAAAAAAAACAAAACTTATCAGATGAGGACCCATGA


 


TTAAGAATTTGTAGCCTTATGGGGAACACAGATCTCTAGTCTCAATTCACAACC


 


TAGTGTTGTGACAAGTTGAAGGCTGGTAGTTAAATTGTTGAGAATATCTCAGA


 


GGGGCCTTTTAGTTTCTCTTGGAAGAAGAGTGAACTCCCAATTCCACAAATTAA


 


AAAATGCAGCTAAATGCTACAAATTGGATTTTTACTAAAAGCACCAATAGGTT


 


CCGTTTGCATTCTGTTACCTACACACCTGTTCTGTTTGTCCCACAGAGGAGGAG


 


GAGGAGGACGATGACGATCTCTTTGATGATCCTCTGCCAGTACCTTTAAGGCA


 


CAAGGTTCCAAATCAGCAGACTCTTCACTCTGAGGTACTTCCCATGACTGCTCT


 


ACCACAAGACCAGCCTGAAAAACAGACAGAAAGCACAGAATGCTTCAAAGCA


 


GAGAGTATGCCAACATGTCTCTGGGCAAACTTCGTAGATTGTGAAGAATCCAA


 


TAGTGAAAGTGAAGAATTAGAAATCACGGCTCCAGCTCAAGGAGACACGAGT


 


CCTGTCCCCCATCACCAGCAGAAGGCTGAAGGGGAAGTACCACAGTGGGAAG


 


TGTTCTTTAAAAGAAATGATGAAATCACAGATGACTGTTTGGAAAACCTTCCGT


 


CCTCCACAGAGGCAGGGGGCTCTCAGTCCCCAAAGCTTTTCAGTGACTCTGAT


 


GGGGAATCAACTCACATTTCTTCCCAGACTTCTTCTCAGTCAACACACATATCA


 


GAACAAGGAAGTCAAGGCTGGGACAGCCAATCAGACACTGTTTTGTTATCTTC


 


CCAAGAGAGAAAAAGTGGGGATATTACCTCCTTGAACAAAGGTGGCTCTAGAC


 


CAGAAATCAAAGAGAATATTCCCATCCTTCAGATGGAACAAAATGTATTTTGC


 


CCGAAGGATACTTACTCTGATTTGAAAGGCAGAGATCAAGATATAAACACACT


 


TCCCAGTGCTAGAGAAACAACTACTCTGAGCAGTGGGAAACACATGCCTCAGG


 


AGAAAAGGCCGCTAAACTGTAACAGTAACACAGATTCACAAGGCTCCTCTGAC


 


TTTGAAATTCCCTCCACTCCAGAAGCTGAGCTACCTCAACAAGAGCATCTGCA


 


ATATTTATACAAGAAGTTGGCAGGAGGAGAGGGTATAGTAATTGAAAAAAGG


 


AAAAGCGCACGTCATTCTAGAGCAACCACTAAAAAACCTACACAAACAGGTA


 


ATAGTCAGACTCCTAATAGATGAGTTCAAATGGAGTACTTAAAAATGTTCATAT


 


AACCTAAAAGGCAGCTCTAAAAGGGGAAACAATAGGACCAAAAAATACAAAG


 


AAAACAAGAATGGGAGAACTCAGTACAAACATATCAGTAATTACATCAAATG


 


AAAATAGGGGAAAAACCATAAGCCAACTATATATTGTCTATAGGAAACTGGCT


 


TCAAGACTTGGGCAGGTTTACTGGTGAAAGGATGGAAACCTTCACCACATAAT


 


AAACATGAAAGATGGAGGGGCTATATTACTAGTGATAAAAGGTCCAGTTCACT


 


GAGACATAAACCCAAATGAGTATGTACTGGACAACAGCCACACATACAGAGC


 


AAAAACAACTGAAAGGAGAAAGATAAACTAAAGCACAATTACAGCTGCAACA


 


GAATTAGTCTACAGGAAATCAGCAAGGAGACGGAAGAACTGAACAGCACTAT


 


CAACCAACCAGATCTTCACAGAAGACACCATCTAGCACAGAATACACACTTTT


 


CAAGTTCACAGAATACAACATTCACCAAGATAGACCATATCCTGGTTCATAAA


 


AACTTGGATCTA

## Claims

1. A transgenic porcine subject whose genome comprises a transgene comprising the nucleotide sequence of SEQ ID NO: 7 or 9, wherein one allele of a genomic Artemis gene is functionally disrupted by said transgene, wherein said disruption comprises a single nucleotide polymorphism selected from the group consisting of a G to A substitution as set forth at position 615 of SEQ ID NO:7 and a G to A substitution as set forth at position 116 of SEQ ID NO:9, wherein said transgenic porcine subject exhibits severe combined immunodeficiency disease (SCID) relative to a wild type porcine subject, said SCID characterized by decreased numbers of T and B lymphocytes, circulating antibody, and significantly abnormal thymus and lymph nodes.

2. A porcine semen comprising isolated sperm cells from the transgenic porcine subject of claim 1, wherein the genome of the isolated sperm cells comprises a transgene comprising the nucleotide sequence of SEQ ID NO: 7 or 9, wherein one allele of a genomic Artemis gene is functionally disrupted by said transgene, wherein said disruption comprises a single nucleotide polymorphism selected from the group consisting of a G to A substitution as set forth at position 615 of SEQ ID NO:7 and a G to A substitution as set forth at position 116 of SEQ ID NO:9, wherein said transgenic porcine subject exhibits severe combined immunodeficiency disease (SCID) relative to a wild type porcine subject, said SCID characterized by decreased numbers of T and B lymphocytes, circulating antibody, and significantly abnormal thymus and lymph nodes.

3. A transgenic porcine embryo obtained by fertilizing an oocyte with an isolated sperm cell from the transgenic porcine subject of claim 1, wherein the genome of the isolated sperm cell comprises a transgene comprising the nucleotide sequence of SEQ ID NO: 7 or 9, wherein one allele of a genomic Artemis gene is functionally disrupted by said transgene, wherein said disruption comprises a single nucleotide polymorphism selected from the group consisting of a G to A substitution as set forth at position 615 of SEQ ID NO:7 and a G to A substitution as set forth at position 116 of SEQ ID NO:9, wherein said transgenic porcine subject exhibits severe combined immunodeficiency disease (SCID) relative to a wild type porcine subject, said SCID characterized by decreased numbers of T and B lymphocytes, circulating antibody, and significantly abnormal thymus and lymph nodes.