# Patent

## Title
Porcine pancreatic amylase gene promoter and transgenic pigs expressing heterologous digestive enzymes

## Abstract

The present invention relates to a novel promoter and its use in driving expression of foreign genes in transgenic animals (especially pigs). Accordingly, the present invention provides a method for producing transgenic animals harboring heterologous genes regulated by the promoter of the present invention.

## Background
FIELD OF THE INVENTION
The present invention relates generally to the expression of heterologous proteins in animals and to the production of transgenic animals.
BACKGROUND OF THE INVENTION
Animal husbandry constitutes the biggest portion of agricultural production in Taiwan, and brings handsome and stable income to Taiwanese farmers. However, fecal waste produced by livestock in large amount is not properly disposed and thus results in environmental pollution. According to the statistical information from the Council of Agriculture, Executive Yuan, Taiwan, the headcount of farmed hogs was 6,794,000 by the end of the year 2002. Assuming that each hog produces 6 Kg of fecal waste per day, there will be about 15,000,000 metric tons of hog fecal waste per year, plus the fecal waste from poultry. The serious environmental pollution resulting from such a large amount of fecal waste has become the major problem in Taiwanese animal husbandry. How to reduce the production of fecal waste without jeopardizing farmers' income is an important issue.
In addition to the aforementioned environmental issue, Taiwanese animal husbandry has to compete with imported livestock products since Taiwan joined the World Trade Organization (WTO) in 2002. How to reduce the production cost while enhancing the production rate has become another important issue. Generally speaking, the cost of feed constitutes 60 to 70% of the total production cost. Therefore, the key to reduce the production cost is to improve feed utilization. Effective ways include utilizing biotechnology to develop feed additives for enhancing the digestion of feed, and breeding livestock animals able to digest cellulose to reduce fecal production.
The quantity and quality of animal feces depend on the digestion and utilization of the components of feed by the animal. Generally speaking, livestock animals do not have the ability to synthesize digestive enzymes such as cellulases and phytases, and thus cannot effectively digest the cellulose and plant phosphorus in the feed. Such inability affects feed utilization and results in a waste of feed. In addition, the undigested nutrients are excreted from the animals and become a cause of environmental pollution. A common method for enhancing utilization of the nutrients in feed by animals is to supplement the feed with certain digestive enzymes produced on large scale by microorganisms. Recently, the cDNAs of several digestive enzymes have been screened out from fungal and bacterial cDNA libraries, and the enzymes have been produced on large scale by E. coli (Ye et al., 2001, Inter. J. Biochem. Biol. 33:87-94; and Zhang et al., 1998, Biotechnol. Lett. 20: 1001-1005; both hereby incorporated herein by reference). However, the production cost of this method is high. In addition, the supplementary digestive enzymes are usually destroyed in the animal's digestive tract by the native enzymes or gastric acid and thus cannot carry out their function.
Since the 1980s, the generation of transgenic animals not only allows us to study gene functions in vivo, but also provides a new way to improve the genetics of livestock animals. In the conventional breeding method, it takes tens of or even over a hundred years to improve the genetics of livestock animals. However, utilizing the techniques of genetic transformation, the same improvement can be achieved within one generation. In 1985, Hammer et al. first published their results in transgenic pigs, sheep and rabbits (Hammer et al., 1985, Nature 315:680-683; hereby incorporated herein by reference). Afterwards, researchers have utilized genes encoding growth factors from various animals (including cattle, mice and humans) to generate transgenic pigs carrying heterologous growth factor genes, hoping to increase lean meat in pigs and to shorten rearing time (Pursel and Rexroad, 1993, J. Anim. Sci. 71:10-19; hereby incorporated herein by reference). For example, Pursel et al. have constructed several heterologous growth factor genes under the control of the mouse metallothionein-I promoter (Pursel et al., 1989, Science 244:1281-1288; hereby incorporated herein by reference), and thereby the transcription of the heterologous genes can be induced and regulated effectively by the addition of trace amounts of zinc in the feed.
As known in the art, manipulation of animals (such as pigs) to alter and/or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in animal tissues. Such genetic manipulation therefore relies on the availability of means to drive and to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in animals and which regulate gene expression so as to give the desired effect(s) in the transgenic animal. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, animal or environment.
There is a continuing need in the art for high-level expression promoters, as well as promoters which are spatially defined in their expression patterns.
BRIEF SUMMARY OF THE INVENTION
In the present invention, a novel porcine pancreatic amylase gene promoter is operatively linked to either a fungal cellulase gene or a bacterial phytase gene to form an expression cassette, which is utilized in the generation of transgenic mice and pigs.
Accordingly, in one aspect, the present invention provides an isolated porcine pancreatic amylase gene promoter. In a preferred embodiment, the promoter has a nucleotide sequence as set forth in SEQ ID NO: 1.
In another aspect, the present invention provides a recombinant DNA construct comprising the porcine pancreatic amylase gene promoter of the present invention operatively linked to a nucleotide sequence of interest. In a preferred embodiment, the nucleotide sequence of interest codes for a digestive enzyme, such as a phytase, a cellulase, a glucanase and a xylanase.
In a further aspect, the present invention provides a method for producing a transgenic animal comprising the steps of providing an animal embryo and introducing into the animal embryo a transgene comprising the porcine pancreatic amylase gene promoter of the present invention operatively linked to a nucleotide sequence of interest, thereby transforming the embryo with the transgene.
In yet another aspect, the present invention provides a transgenic animal whose genome contains a transgene comprising a heterologous nucleotide sequence operatively linked to the promoter of the present invention. In a preferred embodiment, the transgenic animal is a pig.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
In the drawings:
FIG. 1 shows fluorescence microscopic images of AR-42J rat pancreatic tumor cells transfected with 412 pAMY-phrGFP or 1216 pAMY-phrGFP vectors. Panel A1 is the image of 1216 pAMY-phrGFP-transfected AR-42J cells under bright field. Panel A2 is the image of 1216 pAMY-phrGFP-transfected AR-42J cells under dark field. Panel B1 is the image of 412 pAMY-phrGFP-transfected AR-42J cells under bright field. Panel B2 is the image of 412 pAMY-phrGFP-transfected AR-42J cells under dark field.
FIG. 2 shows the results of PCR verification of transgenic mice harboring the cellulase transgene. “M” denotes markers; “+” denotes positive control; “−” denotes negative control; and “W” denotes wildtype. The numbers correspond to the identification numbers of the transgenic mice.
FIG. 3 shows the results of PCR verification of transgenic mice harboring the phytase transgene. “M” denotes markers; “+” denotes positive control; “−” denotes negative control; and “W” denotes wildtype. The numbers correspond to the identification numbers of the transgenic mice.
FIGS. 4A and 4B show the results of PCR verification of transgenic pigs harboring the cellulase transgene. “M” denotes markers; “+” denotes positive control; “−” denotes negative control; and “W” denotes wildtype. The numbers correspond to the identification numbers of the transgenic pigs.
FIG. 5 shows the results of PCR verification of transgenic pigs harboring the phytase transgene. “M” denotes markers; “+” denotes positive control; “−” denotes negative control; and “W” denotes wildtype. The numbers correspond to the identification numbers of the transgenic pigs.
FIG. 6 shows the results of Southern Blot analysis of transgenic pigs harboring the cellulase transgene, wherein the porcine genomic DNA was digested with Afl II. “M” denotes markers; “Pc” denotes positive control; and “Nc” denotes negative control. The numbers correspond to the identification numbers of the transgenic pigs.
FIG. 7 shows the results of Southern Blot analysis of transgenic pigs harboring the cellulase transgene, wherein the porcine genomic DNA was double digested with Hind III and HaeII. “M” denotes markers; “Pc” denotes positive control; and “Nc” denotes negative control. The numbers correspond to the identification numbers of the transgenic pigs.
FIG. 8 shows the results of Southern Blot analysis of transgenic pigs harboring the phytase transgene, wherein the porcine genomic DNA was digested with Drd I. “M” denotes markers; “Pc” denotes positive control; and “Nc” denotes negative control. The numbers correspond to the identification numbers of the transgenic pigs.
FIG. 9 shows the results of Southern Blot analysis of transgenic pigs harboring the phytase transgene, wherein the porcine genomic DNA was double digested with Hind III and ApaL I. “M” denotes markers; “Pc” denotes positive control; and “Nc” denotes negative control. The numbers correspond to the identification numbers of the transgenic pigs.



DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is directed to a novel promoter sequence of the porcine pancreatic amylase gene and uses thereof in the expression of recombinant genes and production of transgenic animals. The promoter of the present invention permits space and time-determined expression of transgenes in a transgenic animal.
In accordance with an embodiment of the present invention, there is provided an isolated porcine pancreatic amylase gene promoter.
As used herein, the term “isolated” refers to material, such as a nucleic acid, which is 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.
As used herein, the term “promoter” refers to an untranslated DNA sequence upstream of a coding region that contains the binding site for an RNA polymerase and initiates transcription of the coding region. The promoter region may also include other elements that act as regulators of gene expression.
In a preferred embodiment, the promoter of the present invention has the nucleotide sequence of SEQ ID NO: 1.
In accordance with another embodiment of the present invention, there is provided a recombinant DNA construct comprising the porcine pancreatic amylase gene promoter of the present invention operatively linked to a nucleotide sequence of interest.
In a preferred embodiment, the recombinant DNA construct is an expression vector comprising an expression cassette. As used herein, the term “expression cassette” refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a nucleotide sequence of interest in a host cell. The expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the expression cassette portion of an expression vector includes, among other sequences, a nucleotide sequence to be transcribed, and a promoter. In the present invention, the expression cassette can also be referred to as a “transgene” when used to produce transgenic animals.
As used herein, “operatively linked” includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the second sequence. Generally, “operatively 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.
According to the present invention, the nucleotide sequence of interest under the control of the promoter can be any nucleic acid form. Correspondingly they can be coding nucleic acids or structural or functional nucleic acids. The term “coding nucleic acid” is understood to mean more particularly a nucleic acid coding for a peptide or protein. The peptide or protein can e.g. be a structural protein or a peptide or protein having enzymatic activity. A “structural nucleic acid” is more particularly understood to mean a nucleic acid leading to the formation of complexes, particularly with other molecules. It can inter alia be an rRNA and in particular an antisense nucleic acid. A “functional nucleic acid” is more particularly understood to mean a nucleic acid, which exerts a specific action on a system, particularly a biological system. Such a specific action can e.g. be the aiding or inhibiting of translation or transcription. An example of a functional nucleic acid is an antisense nucleic acid.
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise.
In a preferred embodiment, the nucleotide sequence of interest under the control of the promoter is a coding nucleic acid. More preferably, the coding nucleic acid codes for a digestive enzyme, such as a phytase, a cellulase, a glucanase or a xylanase.
In accordance with a further embodiment of the present invention, there is provided a method for producing a transgenic animal comprising the steps of providing an animal embryo and introducing into the animal embryo a transgene comprising the porcine pancreatic amylase gene promoter of the present invention operatively linked to a nucleotide sequence of interest, thereby transforming the embryo with the transgene.
According to the present invention, the transgenic animal is preferably a mammal, more preferably livestock such as cattle, sheep or pigs. In a preferred embodiment, the transgenic animal of the present invention is a pig.
Generation of transgenic animals of the present invention is carried out conventionally by techniques well known in the art. There are a number of techniques that permit the introduction of genetic material (such as a transgene) into animals to be transformed, including the viral infection technique; the sperm mediated gene transfer (SMGT) technique; the embryonic stem cell technique; the nuclear transfer technique; and the pronuclear microinjection technique. Among them, the most commonly used technique is the pronuclear microinjection technique, which comprises direct injection of the transgene into the male pronucleus of fertilized eggs, resulting in the random integration into one locus of a varying number of copies, usually in a head to tail array. The injected eggs are then re-transferred into the uteri of pseudo-pregnant recipient mothers. Some of the resulting offspring may have one or several copies of the transgene integrated into their genomes, usually in one integration site. An advantage of the pronuclear microinjection technique is that the transgene would be stably integrated into the germline of transgenic animals so that it will be passed to their offspring.
According to a preferred embodiment of the present invention, the transgene is introduced into the embryo by pronuclear microinjection.
Preferably, prior to the introduction of the transgene into the embryo, it is removed from the vector portion by restriction enzyme digestion, for example by using restriction sites in the vector that flank the transgene. For cloning purposes, the transgene is generally inserted into an expression vector, such as the mammalian expression vector pCR® 3.1 (Invitrogen Corporation), prior to the introduction into the embryo. In general, the transgenic DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
In accordance with yet another embodiment of the present invention, there is provided a transgenic animal whose genome contains a transgene comprising a heterologous nucleotide sequence operatively linked to the promoter of the present invention. In a preferred embodiment, the transgenic animal is a pig.
As used herein, the term “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 engineered from its native form in composition and/or genomic locus by deliberate human intervention. A heterologous protein may originate from a foreign species or, if from the same species, is substantially engineered from its original form by deliberate human intervention.
Preferably, the transgenic animal is generated by introduction of a transgene into an embryo by pronuclear microinjection, insertion of the embryo into a surrogate mother, and allowing the embryo to develop to term.
The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.
Example 1 Cloning of Porcine Pancreatic Amylase Gene Promoter
Darnis et al. has obtained the nearly full-length cDNA sequence of porcine pancreatic a-amylase gene (GenBank Accession No. AF064742) from a cDNA library constructed with the mRNA isolated from pancreatic tissues of pigs (Darnis et al., 1999, Biochem Biophys Acta. 1430:281-289; hereby incorporated herein by reference). The cDNA sequence has a length of 1,536 bp, and encodes a protein of 496 amino acids with a signal peptide of 15 amino acids. Based on the cDNA sequence, the promoter was cloned utilizing GenomeWalkerTM Kit (Clontech Laboratories, Inc., Mountain View, Calif., USA) according to the manufacturer's instructions.
First, the genomic DNA isolated from porcine blood was digested at 37° C. by the four restriction enzymes (Dra I, EcoR V, Pvu II, and Stu I) provided in the kit. After 16 hours, the digested genomic DNA was precipitated by ethanol and resuspended. The resuspended digested genomic DNA was then ligated to the adaptor provided in the kit at 16° C. for 16 hours.
In addition to the two “adaptor primers” (AP1 and AP2) provided in the kit, three primers, GSP1, GSP2 and GSP3 were designed based on the sequence at the 5′ end of the porcine pancreatic amylase gene (see Table 1). The first PCR was conducted utilizing the above-derived digested genomic DNA as a template and GSP3 and AP1 as primers. The second PCR was conducted utilizing the products of the first PCR as a template and GSP2 and AP2 as primers. The products of the second PCR were screened by gel electrophoresis, and possible fragments (i.e., those larger than 1 kb) were recovered from the gel and purified.
The selected fragments were cloned into pGEM®-T Easy Vectors (Promega Corporation, Madison, Wis., USA) and the formed vectors were named pGEM-T AMY. The clones were sequenced and two clones, numbered 412 and 1216, were selected.








TABLE 1







Primers Used in Genome Walking










Primer
Sequence
Tm value (° C.)













AP1
GTAATACGACTCACTATAGGGC
59




(SEQ ID NO: 6)


 


AP2
ACTATAGGGCACGCGTGGT
71



(SEQ ID NO: 7)


 


GSP1
GCTGAAAGCAGCAGAAACTTCAT
60.4



(SEQ ID NO: 8)


 


GSP2
ATGAACAATAGACGTTCGTCCAGACT
59.7



(SEQ ID NO: 9)


 


GSP3
TACCGCTCACATTCAAGAGCAATGTCA
62.3



(SEQ ID NO: 10)









Example 2 Activity Test of Porcine Pancreatic Amylase Gene Promoter
To test the activity of the promoter cloned in Example 1, new primers (1216AMY-GFP-NotI-5′ and 1216AMY-GFP-EcoRV-3′) (see Table 2) were designed in order to create an Not I restriction site at the 5′ end and an EcoR V restriction site at the 3′ end of the promoter by PCR. The PCR products were again cloned into pGEM®-T Easy Vectors and digested with the relevant restriction enzymes. The digested fragments were cloned into phrGFP vectors (Stratagene, La Jolla, Calif., USA) and the clones were sequenced. Two clones, 412 pAMY-phrGFP and 1216 pAMY-phrGFP, were obtained.
Rat pancreatic tumor cell line AR-42J (BCRC 60160) was purchased from the Bioresource Collection and Research Center of Food Industry Research and Development Institute, Hsinchu, Taiwan. The cells were cultured on a six-well culture plate, with each well containing 7×104 cells. The cultured cells were transfected with lipofectomine (Invitrogen Corporation, Carlsbad, Calif., USA) and either 412 pAMY-phrGFP or 1216 pAMY-phrGFP vectors. After culturing at 37° C. for 24 hours, green fluorescence was observed using a fluorescent microscope (FIG. 1). These results showed that the cloned promoter was able to drive the expression of heterologous genes (in this case, the coding sequence for green fluorescence protein) in pancreatic cells.








TABLE 2







Primers Used in Constructing 1216pAMY-phrGFP,



1216pAMY-CEL and pAMY-PHY












Tm value



Primer
Sequence
(° C.)





1216AMY-GFP-
GCGGCCGCCTGACATAAGCTGAA
79.5



NotI-5′
(SEQ ID NO: 11)


 


1216AMY-GFP-
GATATCGGCCCAGCAGAACCCAA
76.4


EcoRV-3′
(SEQ ID NO: 12)


 


1216AMY-NheI-5′
GCTAGCCTGACATAAGCTGAACCAA
71.8



(SEQ ID NO: 13)


 


1216AMY-BamHI-3′
GGATCCGGCCCAGCAGAACCCAA
82.0



(SEQ ID NO: 14)


 


CEL-BamHI-5′
GGATCCATTATGAAACCCGAACCA
69.7



(SEQ ID NO: 15)


 


CEL-XhoI-3′
CTCGAGTTATTCCTTTGGTTTTTC
66.6



(SEQ ID NO: 16)


 


PHY-BamHI-5′
GGATCCCAGAGTGAGCCGGAGCT
74.1



(SEQ ID NO: 17)


 


PHY-XhoI-3′
CTCGAGTTACAAACTGCACGCCGGTA
78.1



(SEQ ID NO: 18)









Example 3 Preparation of Transgene Constructs for Microinjection
A cellulase gene (GenBank Accession No. AF053363) from Piromyces rhizinflatus and a phytase gene (GenBank Accession No. AF537219) from an E. coli strain isolated from bovine feces (ATCC 33965) were used to construct the transgenes. cDNAs of both genes were provided by Dr. Kuo-Joan Cheng of Institute of Bioagricultural Science, Academia Sinica, Taipei, Taiwan. The cDNAs of both genes were first amplified by PCR using primer pairs CEL-BamHI-5′+CEL-XhoI-3′ (for the cellulase gene) and PHY-BamHI5′+PHY-XhoI-3′ (for the phytase gene) (see Table 2) and cloned into pGEM®-T Easy Vectors. The promoter cloned in Example 1 was also amplified by PCR using primer pair 1216AMY-NheI-5′+1216AMY-BamHI-3′ (see Table 2) and the PCR products were also cloned into pGEM®-T Easy Vectors.
The promoter (digested with Nhe I and BamH I) and either the cellulase or phytage gene (digested with BamH I and Xho I) were cloned into the mammalian expression vector pCR® 3.1 (Invitrogene Corporation) to form the vector pAMY-CEL or pAMY-PHY. After mass production of the vectors, a transgene (SEQ ID NO:2) containing the promoter, a signal peptide, a structural gene expressing a polypeptide having the sequence of SEQ ID NO:3 and a BGH poly A tail and a transgene (SEQ ID NO:4) containing the promoter, the signal peptide, a structural gene expressing a polypeptide having the sequence of SEQ ID NO:5, and a BGH poly A tail were excised from the vectors by restriction enzyme digestion. The excised transgenes were recovered trice with QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, Calif., USA) and subjected to CsCl density gradient centrifugation (CsCl, 1 gm/ml; ethylene bromide (EthBr), 24 μl; DNA, 100-200 μg, 0.02% Triton-X100) at 100,000 rpm for 6 hrs. After centrifugation, the band containing linear DNA was extracted under UV light and mixed with saturated butanol aqueous solution to remove EtBr. The DNA was then dialyzed with TE buffer (pH 8.0) for 24 hours to remove CsCl, precipitated with ethanol, and redissolved in TE buffer (pH 7.4). After the concentration and quality of the DNA were determined by gel electrophoresis and a spectrophotometer, the DNA was diluted to 3 ng/μl and stored under −20° C.
Example 4 Production of Transgenic Mice
The ICR female mice used as embryo donors and recipients were purchased from National Taiwan University Animal Center (Taipei, Taiwan). The present experiment was conducted on 6 to 7 week old, sexually mature female mice.
Fertilized mouse embryos of the pronucleus stage were flushed out and recovered from the oviducts of superovulated female mice. Ten to twenty embryos to be microinjected were placed into a microdrop of M2 buffer in the center of a depression microscope slide. The microdrop was covered with heavy mineral oil to prevent evaporation of the buffer. Microinjection was performed using a differential interference contrast microscope (Axiovert 10, Zeiss, Germany) with Narashigi NT-8 micromanipulators (Narashigi, Japan). The holding pipette for stabilizing the embryo and the injection pipette for DNA injection were prepared as described in Wu et al., 1995, J. Chin. Soc. Anim. Sci. 24:181-189 (hereby incorporated herein by reference). About 2 pl of DNA solution containing 3 to 5 ng/μl of the transgene constructs prepared in Example 3 were injected into the male pronucleus of each embryo.
Embryos surviving the microinjection process as judged by morphological observation (i.e., those retaining dense deutoplasm and intact appearance) were implanted into recipient females made pseudo-pregnant by mating with vasectomized males. About 10 to 20 microinjected mouse embryos were transferred into each of the two oviducts of pseudopregnant females. Embryos were allowed to come to term and the newborn mice were analyzed for the presence of the transgenes by PCR as described below.
In the present study, 267 ICR mouse embryos were microinjected with the transgenes, among which 245 surviving embryos were implanted into the oviducts of recipient female mice (the loss rate for microinjected embryos was 8.2%). A total of 63 mouse pups were born, meaning that the survival rate for mouse embryos was 25.7%.
Example 5 Analysis of Transgenic Mouse Genomic DNA
Among the 63 potential transgenic mouse pups obtained in Example 4, only 57 have successfully grown up to weaning age. Using tissue samples removed from the tails of the 57 surviving pups, genomic DNA was obtained by the following procedures.
The tissue sample was cut into tiny pieces and mixed with a solution containing 630 μl of tissue lysis buffer (100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 200 mM NaCl), 70 μl of 10% SDS, and 35 μl of 100 mg/ml Proteinase K. The mixture was incubated in a 55° C. water bath for 12 to 16 hours, and then centrifuged at 12,000× rpm for 5 minutes. The supernatant from centrifugation was extracted twice with phenol/chloroform (1:1), and then centrifuged at 12,000× rpm again for 5 minutes to obtain two layers. DNA was precipitated from the upper layer with absolute ethanol, washed with 70% ethanol, and redissolved in autoclaved distilled and deionized H2O.
PCR reactions on the genomic DNA samples obtained above were carried out conventionally. Primer pairs were designed based on the sequences of the porcine pancreatic amylase gene promoter, the junction between the promoter and the structural gene, the structural gene, and the BGH poly A tail (Table 3). To primarily examine if a potential transgenic mouse carries the desired transgene, the genomic DNA sample was subjected to PCR (reaction volume=20 μl) using either primer pair 1216-pF+CEL-R (for the cellulase gene) or 1216-pF+PHY-R (for the phytase gene). For the cellulase gene, the reaction conditions were: (1) 94° C., 2 mins; (2) 94° C., 30 secs; 65° C., 30 secs; and 72° C., 2 mins; 35 cycles; (3) 72° C., 7 mins; and (4) 4° C., termination. For the phytase gene, the reaction conditions were: (1) 94° C., 2 mins; (2) 94° C., 30 secs; 67° C., 30 secs; and 72° C., 2 mins; 35 cycles; (3) 72° C., 7 mins; and (4) 4° C., termination.
The results of the PCR analysis are shown in FIGS. 2 and 3. As can be seen from the results, pups No. 4, 29, 33 and 48 carry the cellulase transgene (FIG. 2), while pups No. 1, 20, 29, 33, 47 and 48 carry the phytase transgene (FIG. 3), which means that pups No. 29, 33 and 48 carry both transgenes. The identities of the transgenes were further confirmed by PCR using primer pair 1216C-jF+1216C-jR or 1216P-jF+1216P-jR.








TABLE 3







Primers Used in Transgenic Animal DNA Analysis














Tm






value
PCR


Primer
Sequence
(° C.)
Product





1216-pF
AGGGACCGAAGGAGAGTGTT
64.1
314 bp




(SEQ ID NO: 19)


 


1216-pR
ACCATTCTTGCTCTGCTGTGA
64.6



(SEQ ID NO: 20)


 


1216C-jF
ACCGTTGACAACCTCAGAGCA
66.8
404 bp



(SEQ ID NO: 21)


 


1216C-jR
TCGTTCACCAAAGTGTCCAGA
65.2



(SEQ ID NO: 22)


 


1216C-aF
CATTGTTGCAGCCTTACAA
59.0
262 bp



(SEQ ID NO: 23)


 


1216C-aR
ACTCAGACAATGCGATGCA
60.8



(SEQ ID NO: 24)


 


1216P-jF
ACCGTTGACAACCTCAGAGCAA
68.8
350 bp



(SEQ ID NO: 25)


 


1216P-jR
ACGCTCGTCGACATCAGCAATA
70.0



(SEQ ID NO: 26)


 


1216P-aF
TCGGCTAAGCGATAACAG
58.4
291 bp



(SEQ ID NO: 27)


 


1216P-aR
ACAACAGATGGCTGGCAACT
64.1



(SEQ ID NO: 28)


 


CEL-R
GGATCCTTATTCCTTTGGTTTTTC
53.8



(SEQ ID NO: 29)


 


PHY-R
GGATCCTTACAAACTGCACGCCGG
64.5



(SEQ ID NO: 30)


 


CEL-Hyb-3′
TCCGTTCCATTCAACTGGTG
65.2



(SEQ ID NO: 31)


 


PHY-Hyb-3′
TCAGTCACGTTCGCGTTATCT
65.0



(SEQ ID NO: 32)









Example 6 Production of Transgenic Pigs
Sexually mature crossbred gilts (more than 6 month old) were used in the present study as embryo donors and recipients. Superovulated donor gilts were artificially inseminated and fertilized embryos were surgically recovered from their oviducts. The embryos were centrifuged at 12,000× g for 10 minutes at 25° C. in order to show the pronuclei (the cytoplasm of porcine embryos was opaque with lipid and pronuclei were invisible before centrifugation).
Microinjection of the embryos was performed as described above in Example 4. Embryos surviving the microinjection process as judged by morphological observation were implanted into oestrous recipient gilts. About 10 to 20 microinjected porcine embryos were transferred into each of the two oviducts of recipient gilts. Embryos were allowed to come to term and the newborn pigs were analyzed for the presence of the transgenes by PCR and Southern blot as described below.
In the present study, 145 porcine embryos were microinjected with the transgenes, among which 138 surviving embryos were implanted into the oviducts of 6 recipient gilts. Among the 6 recipients, 2 were pregnant and gave birth to a total of 13 piglets.
Example 7 Analysis of Transgenic Pig Genomic DNA
Genomic DNA was obtained from tissue samples removed from the ears of the 13 potential transgenic piglets by the same procedures as described in Example 5. PCR was performed on the genomic DNA samples using one of the following primer pairs: 1216-pF+1216C-aR (for full-length cellulase transgene), 1216-pF+1216P-aR (for full-length phytase transgene), 1216C-jF+1216C-jR (for promoter-cellulase junction) and 1216P-jF+1216P-jR (for promoter-phytase junction) (see Table 3).
The results of the PCR analysis are shown in FIGS. 4 and 5. As can be seen from the results, piglets No. 29-3, 29-7, 29-8, (FIG. 4A) and 13-2 and 13-5 (FIG. 4B) carry the cellulase transgene, while piglets No. 29-7 and 29-8 carry the phytase transgene (FIG. 5), which means that piglets No. 29-7 and 29-8 carry both transgenes. Among the 5 transgenic piglets, piglet No. 13-2 died of bacterial infection 10 days after birth.
Southern blotting was performed on the genomic DNA of the 5 transgenic piglets in order to understand the level of integration of the transgenes, using PCR products of primer pairs 1216C-aF+1216C-aR, 1216P-aF+1216P-aR, CEL-BamHI-5′+CEL-Hyb-3′ and PHY-BamHI-5′+PHY-Hyb-3′ (see Tables 2 and 3) as probes. Recovered and purified PCR products were first denatured in boiling water and placed on ice. Radioactive labeling of the PCR products was carried out with RediprimeTM II DNA Labeling System (GE Healthcare UK Ltd, UK) according to the manufacturer's instructions. Southern Blotting was performed essentially as described in Koetsier et al., 1993, Biotechniques 15(2):260-2 (hereby incorporated herein by reference). The procedures employed are outlined below.
Genomic DNA of the 5 transgenic piglets digested with relevant restriction enzymes was subjected to 0.8% agarose gel electrophoresis (50V, 4-6 hrs) and depurination with 0.25 N HCl for 15 minutes. After the Bromophenol Blue dye turned yellow, the gel slab was incubated in a denaturing solution containing 1.5 M NaCl and 0.4 N NaOH. After blotting for 2.5 to 4 hours, the nylon membrane (HybondTM-N+, GE Healthcare UK Ltd) with DNA blots was placed into a 2×SSC solution for neutralizing the reaction. For pre-hybridization, the membrane was incubated in a pre-hybridization solution (2×SSC, 1% SDS, 0.5% fat-free milk powder and 0.75 mg/ml denatured salmon sperm DNA) at 66° C. for 2 to 5 hours. For hybridization, the membrane was moved into a hybridization solution (2×SSC, 1% SDS, 0.5% fat-free milk powder, 0.5 mg/ml denatured salmon sperm DNA and 10% dextran sulfate) containing the radioactive labeled probes, and incubated at 66° C. for 16 to 24 hours. After hybridization, the membrane was twice washed at 55° C. for 20 minutes with a solution containing 0.1% SDS and 0.1×SSC. Finally, the membrane was air dried and analyzeded by a Bio-imaging Analyzer (BAS-1500, FUJIFILM, Japan).
The results of the Southern Blot analysis are shown in FIGS. 6 to 9. In FIG. 6, the porcine genomic DNA was digested with Afl II and the probe was the PCR product of primer pair 1216C-aF+1216C-aR. The image shows that all of the 5 transgenic piglets carry the cellulase transgene, but integrated copy numbers may be larger in piglets No. 13-2 and 29-8 in view of the stronger hybridization signals for the two. In FIG. 7, the porcine genomic DNA was double digested with Hind III and Hae II and the probe was the PCR product of primer pair CEL-BamHI-5′+CEL-Hyb-3′. The image shows that except for piglet No. 13-2 (whose genomic DNA was damaged), the other four piglets all have the 3,726-kb fragment same as the positive control, meaning that they all carry the cellulase transgene but with different integration levels. In FIG. 8, the porcine genomic DNA was digested with Drd I and the probe was the PCR product of primer pair 1216P-aF+1216P-aR. The image shows that piglet No. 29-8 carries the phytase transgene, but the restriction digestion for piglet No. 29-7 seems insufficient. In FIG. 9, the porcine genomic DNA was double digested with Hind III and ApaL I and the probe was the PCR product of primer pair PHY-BamHI-5′+PHY-Hyb-3′. As can be seen from the image, only piglet No. 29-8 has the 3,892-kb fragment same as the positive control. However, piglet No. 29-7 has a fragment near the 8 kb position, which is believed to represent the undigested phytase transgene.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

## Claims

1. An isolated DNA promoter comprising the nucleotide sequence of SEQ ID NO:1.

2. A recombinant DNA construct comprising the promoter according to claim 1 operatively linked to a nucleotide sequence of interest.

3. The recombinant DNA construct according to claim 2, wherein the nucleotide sequence of interest is a coding sequence.

4. The recombinant DNA construct according to claim 3, wherein the nucleotide sequence of interest encodes a digestive enzyme.

5. The recombinant DNA construct according to claim 4, wherein the digestive enzyme is selected from the group consisting of a phytases, a cellulases, a glucanase and a xylanase.

6. The recombinant DNA construct according to claim 2, comprising the nucleotide sequence of SEQ ID NO: 2.

7. A method for producing a transgenic pig that exhibits enhanced digestion of cellulose, comprising the steps of (a) providing a pig embryo, (b) introducing into the pig embryo by pronuclear microinjection a transgene including a promoter comprising the nucleotide sequence of SEQ ID NO:1 operatively linked to the nucleotide sequence of SEQ ID NO:2, (c) inserting the pig embryo into a pseudopregnant pig, (d) allowing the pig embryo to develop to term, thereby producing a transgenic pig, and (e) identifying the transgenic pig from step (d) whose genome comprises the transgene.

8. A transgenic pig whose genome comprises a transgene including a promoter comprising the nucleotide sequence of SEQ ID NO:1 operatively linked to the nucleotide sequence of SEQ ID NO:2, wherein the transgenic pig exhibits enhanced digestion of cellulose.

9. The recombinant DNA construct according to claim 6, further comprising the nucleotide sequence of SEQ ID NO: 4.

10. The method according to claim 7, wherein the transgene further comprises the nucleotide sequence of SEQ ID NO:4.

11. The transgenic pig according to claim 8, wherein the transgene further comprises the nucleotide sequence of SEQ ID NO: 4.