Mutant RAG-1 deficient animals having no mature B and T lymphocytes

Immunodeficient animals are generated by introducing a mutation in RAG-1 into the germline of the animals via gene targeting in embryonic stem cells. The production of mutant RAG-1 deficient mice is detailed. RAG-1 deficient mice have no mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. To date, these mice do not have mature B and T lymphocytes, nor do they express immunoglobulin or T cell receptors. The same strategy can be applied to the generation of other RAG-1 deficient animals, such as rabbits, rats, and pigs, using known techniques. These animals are all useful for the same general purposes as the scid mice, for example, cultivation of human lymphocytes for expression of human immunoglobulin. Other uses include the establishment of continuous lymphoid cell lines and, by crossbreeding with other lines of animals, the establishment of animals developing tumors for use in studying tumor cell developments and treatments.

BACKGROUND OF THE INVENTION 
This invention is generally in the area of immunodeficient mutant animals 
and methods of use thereof. 
Assembly of immunoglobulin (Ig) heavy and light chain genes and of .alpha. 
and .beta. chain genes of the T cell receptor (TCR) is mediated to a large 
extent in developing lymphocytes by somatic recombination, in which widely 
separated gene segments are joined to form a complete variable region, a 
process known as V(D)J recombination, described by Tonegawa, S. Nature 
302, 575-581 (1983). The genes for the antigen receptors are produced 
exclusively in lymphocytes through this recombinational process, which 
joins variable (V), diversity (D) and joining (J) gene segments. V(D)J 
recombination occurs at seven different loci: the immunoglobulin (Ig) 
heavy chain, kappa and lambda light chain loci in B lymphocytes (Tonegawa, 
1983) and the T cell receptor (TCR) alpha, beta, gamma and delta chain 
loci in T lymphocytes (Davis, M. M. and Bjorkman, P. J. Nature 334, 
395-402 (1988)). 
The prevailing model is that a common recombinase is active in precursors 
of both B and T cells (Yancopoulos, G. D. et al., Cell, 44:251-259 
(1986)), and that the sequential recombinations are executed by 
developmentally controlled targeting of the recombinase to the different 
loci (Alt, F. W., et al., Immunol. Rev. 89, 5-30 (1986)). The assembly 
process is tightly regulated, occurring in a preferred temporal order (D 
to J joins occur before V to D, lambda segments rearrange before kappa and 
in a lineage specific manner (loci recombined in T cells are never fully 
rearranged in B cells). Developing B cells and T cells rearrange distinct 
gene segment families in a well-defined temporal order. In developing B 
cells, the heavy chain locus is rearranged before the light chain loci. 
Maki, R. et al., Science, 209:1366-1369 (1980); Perry, R. P. et al., Proc. 
Natl. Acad. Sci., USA, 78:247-251 (1981); Alt, F. et al., Cell, 27:381-390 
(1981); Alt, F. et al., EMBO J., 3:1209-1219 (1984); Reth, M. G. et al., 
Nature, 317:353-355 (1985). In developing T cells, the .beta. chain locus 
is rearranged before the .alpha. chain locus. Raulet, D. H. et al., 
Nature, 314:103-107 (1985); Snodgrass, H. R. et al., Nature, 315:232-233 
(1985); Samelson, L. E. et al., Nature, 315:765-768 (1985). 
The complex mechanisms regulating V(D)J recombination are not well 
understood. Rearrangements are mediated by recombination signal sequences 
(RSSs) that flank all recombinationally competent, V, D and J gene 
segments. These signals are conserved among the different loci and species 
that carry out V(D)J recombination and are functionally interchangeable. 
RSSs, necessary and sufficient to direct recombination, consist of a 
syad-symmetric heptamer, an AT-rich nonamer and an intervening spacer 
region of either 12 or 23 bp. The two spacer lengths define two different 
RSSs and one of each is required for efficient joining to occur. 
A number of activities must be involved in the joining reaction; these 
include the recognition of RSSs, enconucleolytic cleavage at or near the 
signal border, base trimming and addition (the joints of coding sequences 
are imprecise) and ligation of the cleaved ends (Sakano, H. et al. Nature, 
280:288-294 (1979); Max, E. E. et al., Proc. Natl. Acad, Sci., USA, 
76:3450-3454 (1979); Early, P. et al., Cell, 19:981-992 (1980); Sakano, H. 
et al., Nature, 290:562-565 (1981); Davis, M. M., Annu. Rev. Immunol., 
3:537-560 (1985). Gene segments flanked by joining signals with 12 bp 
spacers are joined only to gene segments flanked by joining signals with 
23 bp spacers. Although different sets of genes are rearranged in 
developing B and T cells, exogenously introduced T cell receptor gene 
segments can be efficiently recombined in pre-B cells. This suggests that 
B and T cell lineages use the same recombination machinery, as discussed 
by Yancopoulos, G. D. et al., (1986). 
The cis-acting sequences required for V(D)J recombination have been 
described in detail (Tonegawa, 1983; Hesse et al., Genes Dev. 3, 1053-1061 
(1989). A gene called the recombination activation gene RAG-1 has been 
isolated by virtue of its ability to activate V(D)J recombination in NIH 
3T3 fibroblasts on an artificial recombination substrate carrying 
selectable markers (Schatz and Baltimore, 1988; Schatz, D. G., et al. Cell 
59, 1035-1048 (1989)). A second, structurally unrelated gene called RAG-2 
was later identified in the immediate vicinity of RAG-1 (Oettinger, et 
al., Science 248, 1517-1523 (1990). 
A model has been proposed by Oettinger et al., (1990), in which RAG-1 and 
RAG-2 together are sufficient to induce V(D)J recombination in 
fibroblasts. The expression of both genes is concordant and restricted to 
cell lines displaying V(D)J recombination activity and developing lymphoid 
tissues (Schatz et al., 1989; Oettinger et al., 1990; Boehm, T. and 
Rabbitts, T. Proc. Natl. Acad. Sci. USA (1991); Turka et al., Science 253, 
778-781 (1991). The only reported discordancies in their expression 
patterns are transcription of only RAG-1 in the central nervous system of 
the mouse, reported by Chun, et al., Cell 64, 189-200 (1991), and of only 
RAG-2 in the bursa of Fabricius of the chicken, reported by Carlson, et 
al. Cell 64, 201-208 (1991). 
An animal model first described by Bosma et al., Nature 301, 527-530 
(1983), for severe combined immunodeficiency (the scid mouse) has been 
reported that has been utilized for a number of different studies, 
including expression of human antibodies following injection of human 
lymphocytes into the mice, as described for example by McCune, J. M. Curr. 
Op. Immunol. 3, 2, 224-228 (1991). As reported by Bosma, G. C., et al., J. 
ExP. Med. 167, 1016-1033 (1988), the scid mouse, however, does produce 
some mouse immunoglobulin and some T cells, due to mostly aberrant 
rearrangements. Flow cytometric analysis of lymphoid organs reveals a 
blockade of lymphocyte differentiation at an immature stage, as further 
reviewed by Bosma, M. J. and Carroll, A. M. Annu. Rev. Immunol. 9, 323-350 
(1991). 
It is therefore an object of the present invention to produce an animal 
that is totally unable to produce functional immunoglobulin or T cell 
receptors. 
It is a further object of the present invention to determine whether both 
RAG-1 and RAG-2 are required in vivo for expression of functional 
immunoglobulin and T cell receptors. 
SUMMARY OF THE INVENTION 
Immunodeficient animals are generated by introducing a mutation in RAG-1 
into the germline of the animals via gene targeting in embryonic stem 
cells. In the following description, the production of RAG-1 deficient 
mice is detailed. RAG-1 deficient mice have no mature B and T lymphocytes. 
The arrest of B and T cell differentiation occurs at an early stage and 
correlates with the inability to perform V(D)J recombination. To date, 
these mice do not have mature B and T lymphocytes, nor do they express 
immunoglobulin or T cell receptors. The same strategy can be applied to 
the generation of other RAG-1 deficient animals, such as rabbits, rats, 
and pigs, using known techniques. These animals are all useful for the 
same general purposes as the scid mice, for example, cultivation of human 
lymphocytes for expression of human immunoglobulin. Other uses include the 
establishment of continuous lymphoid cell lines and, by crossbreeding with 
other lines of animals, the establishment of animals developing tumors for 
use in studying tumor cell developments and treatments.

DETAILED DESCRIPTION OF THE INVENTION 
It has been discovered that it is possible to produce a severely combined 
immunodeficient animal by inactivating the RAG-1 or RAG-2 gene in the 
animal. Although initial in vitro evidence was unclear whether both the 
RAG-1 and RAG-2 genes were required for production of immunoglobulin and 
TCR, it has now been determined that both genes must be expressed to yield 
functional recombinases for the animal to be fully immunocompetent. As 
described in detail below with reference to the production and 
characterization of a mutant mouse containing a deletion in the RAG-1 
gene, any animal can be constructed in a similar fashion by inactivation 
of the RAG-1 or RAG-2 gene. There are differences, however, in the two 
genes which require that the two genes be dealt with on an individual 
basis and one cannot extrapolate from one gene to the other. 
As described below, a deletion was introduced into the RAG-1 gene, shown 
below, of pluripotent embryonic stem (ES) cells, as described by Evans, M. 
J., and Kaufmann, M. H. Nature 292, 154-156 (1981), using the targeted 
gene disruption technique of Capecchi, M. R. Science 244, 1288-1292 
(1989), in order to produce mice homozygous for the RAG-1 mutation 
(hereafter called homozygous, mutant or RAG-1 deficient mice, or 
homozygotes) resulting in expression of a non-functional recombinase. 
Although described with specific reference to a mouse, the same techniques 
can be used to produce other animals, or cell lines derived from other 
animals or humans, which are deficient in RAG-1. 
Although the example uses a substantial deletion of the gene to produce a 
non-functional recombinase, any alteration which would make the RAG-1 gene 
encode a non-functional recombinase, or interfere with expression of the 
gene, such as the addition, deletion, substitution, or modification of one 
or more bases of the gene or the sequences controlling its expression, 
could be used. These alterations include insertion of the neo gene, or 
other DNA sequences, into the RAG-1 gene, thereby interrupting the gene, 
frameshift mutations, other deletions in or around the gene, and mutations 
of the RAG-1 promoter, enhancer, or splice sites (donor and acceptor). 
The RAG-1 gene sequence is described in Schatz, et al., Cell 59, 1035-1048 
(1989), the teachings of which are incorporated herein. The nucleotide 
sequence (Sequence I.D. No. 1) and the amino acid sequence (Sequence I.D. 
No. 2) are appended hereto. 
See also Tybulewicz, et al., Cell 65, 1153-1163 (1991) and Adra, et al., 
Gene 60, 65-74 (1987). 
The RAG-1 deficient mice produced in the following example do not have any 
mature B and T lymphocytes. Flow cytometric analysis of lymphoid organs 
reveals a blockade of lymphocyte differentiation at an immature stage, 
similar to the situation of the scid mouse, described by Bosma and Carroll 
(1991). Southern blot analysis of DNA from thymus and bone marrow-derived 
Abelson-transformed cell lines indicates that both Ig and TCR gene loci 
remain in the germline configuration. 
A similar phenotype is described in RAG-2 deficient mice by Shinkal et al., 
Cell (in press, 1992). If either RAG-1 or RAG-2 gene is inactivated, the 
animal does not contain mature lymphocytes. Taken together, these data 
suggest that RAG-1 and RAG-2 are both necessary in vivo to either activate 
or catalyze the V(D)J recombination reaction. 
The availability of the RAG-1 deficient animals facilitates many basic and 
applied research projects. 
The RAG-1 mutant mice are a useful alternative to the scid mouse. In 
addition to leakiness, discussed in more detail below, the scid mouse has 
at least two other drawbacks compared to the RAG-1 mutation: the genetic 
defect has not been characterized and the mutation is known to affect 
other processes such as double-strand break-related DNA-repair, as 
reported by Fulop et al., Nature 347, 479-482 (1990); Hendrickson et al., 
Proc. Natl. Acad. Sci. USA 88, 4061-4065 (1991). The RAG-1 mouse or other 
RAG-1 deficient animals can be used for any application the scid mouse is 
used for. 
Other proposed uses for the RAG-1 deficient, or mutant, animals: 
Lymphopoiesis: 
The rearrangement of antigen receptor genes is thought to be preceded by 
the commitment of hemopoietic progenitor cells first to lymphoid cell 
development and then to the progenitor of the three major lymphocyte 
classes, .alpha..beta. T cells, gamma-delta T cells and B-cells. In RAG-1 
deficient mice, such progenitors could accumulate and be used to establish 
continuous cell lines. Transfection of the cell lines with the intact 
RAG-1 gene can reveal their commitment to one or more of the major 
lymphocyte classes. The lines will be useful to study the effect of the 
known cytokines and to identify putative new factors which regulate the 
growth and differentiation of these cells. The information obtained may be 
exploited to improve the recovery of the immune system after human bone 
marrow transplantation. 
Leukemogenesis or development of lymphomas 
The differentiation arrest of lymphoid progenitor cells in RAG-1 deficient 
mice is expected to lead to the development of leukemias or lymphomas. 
Such tumors have not yet been observed in RAG-1 deficient mice but might 
occur at high frequency if activated oncogenes or tumor suppressor gene 
defects are introduced in these mice by breeding with transgenic mice or 
other mutant mice. Such studies may increase the understanding of lymphoid 
tumor cell development and perhaps lead to new approaches to therapy. 
Transplantation of human hemopoietic cells or tissues 
Various groups have transplanted human blood cells, human progenitor cells 
or fragments of human fetal liver, thymus and lymph nodes to 
immunodeficient SCID or xid/beige/nude mice. Because of the complete 
absence of T- and B-cells, RAG-1 deficient mice are preferable as 
recipients. If necessary, the deficient animals can be additionally 
depleted of NK cells by breeding with beige mice or by treatment with 
anti-asialo GM1 antibodies. There are many potential applications of such 
mice. For example: 
The RAG-1 deficient animals are useful in studies of human hemopoietic 
cells, such as in the identification of progenitor cells including 
lymphoid progenitors and pluripotential stem cells; in the identification 
of new cytokines which play a role in the growth and differentiation of 
human hemopoietic cells; in the analysis of the effect of known cytokines; 
and in the analysis of drugs on human hemopoietic cells. 
The RAG-1 deficient animals are also useful in studies on pathogenetic 
mechanisms in diseases caused by lymphotropic viruses such as human 
immunodeficient virus (HIV), human T lymphotropic virus (HTLV-1) or 
Epstein Barr virus (EBV), and in the course of determining new therapeutic 
approaches. 
The RAG-1 deficient animals are also useful in studies of defense 
mechanisms against microorganisms that cause diseases in immunocompromised 
patients such as cytomegalovirus, pneumocystis carinii or candida. 
The RAG-1 deficient animals should be useful in the preclinical evaluation 
of "gene therapy". Genes may be introduced into hemopoietic progenitor 
cells, preferably into pluripotential stem cells with self-renewal 
capacity from patients with inherited genetic defects. The transfected 
cells can be kept frozen until their suitability for clinical use has been 
demonstrated in the RAG-1 deficient animals. 
EXAMPLE 1 
Generation of the Mutation in RAG-1 in ES Cells and in Mice 
Construction of Targeting Vector 
RAG-1 was cloned by screening a genomic EMBL-3 phage library prepared from 
D3 embryonic stem cells (Gossler et al., Proc. Natl. Acad. Sci. USA 83, 
9065-9069 (1986)) (gift from Alcino Silva) with a probe generated by the 
polymerase chain reaction (PCR) technique (gift from Asa Abeliovich). One 
positive phage out of 1.2 million plaques screened was purified and mapped 
by restriction enzyme analysis. A 9 kb Cla1 and a 6.5 kb BamHI-Cla1 
fragment were subcloned into pBluescript/SK--(Stratagene) and further 
mapped. The targeting vector pPMKO-31 was constructed in a quatrimolecular 
ligation reaction, using a 6 kb ClaI-ApaI fragment upstream of the RAG-1 
coding sequence (ending at position 482), a 1.8 kb fragment containing the 
pgk-neo gene (Adra, et al., 1987) (gift from Michael A. Rudniki) excised 
with ApaI and BamHI, a 1277 bp BglI-MluI fragment containing sequences 
between positions 1837 and 3113, and the plasmid pGEM7 (Promega) cut with 
ClaI and MluI. This targeting vector is designed to delete 1356 
nucleotides of the RAG-1 coding sequence between positions 482 and 1837 
and replace it with the pgk-neo gene, transcribed in the opposite 
orientation. The source of the pgk-neo gene is the plasmid pKJ1, described 
in Tybulewicz, 1991. 
Targeting Experiment 
ES cells were grown on mitomycin C or gamma-irradiated primary embryonic 
fibroblasts, and during the selections on G418-resistant fibroblasts 
isolated from transgenic embryos carrying a neo-resistance gene (Gossler 
et al., 1986). The medium composition was as described in Robertson, E. J. 
"Embryo-derived stem cell lines" in: Teratocarcinomas and embryonic stem 
cells: A practical approach. E. J. Robertson, ed. 71-112 
(Oxford-Washington, D.C.: IRL Press, 1987). About 5.times.10.sup.7 AB1 
(McMahon, A. P., and Bradley, A. Cell 62, 1073-1085 (1991)) embryonic stem 
cells were electroporated with 75 .mu.g of Mlu1 linearized pPMKO-31 using 
a Bio-Rad gene pulser set at 800 Volt and 3 .mu.Farad. Selection was 
initiated 20 hours later at a concentration of 125 to 150 .mu.g/ml active 
concentration of G418 (Gibco). Colonies were picked from day 6 to day 9 of 
selection into 96 or 24 well dishes (Costar) and expanded. Half of a 24 
well was frozen down and the other half used to isolate DNA for Southern 
blot analysis. The colonies were screened individually by cutting genomic 
DNA with Xho1 and BamHI and probing the Southern blots with the external 
probe M-N, a 268 bp fragment containing RAG-1 coding sequence between 
positions 311 and 3380 (Schatz, et al., 1989). 
The targeting vector pPMKO-31 (FIG. 1) was constructed from a RAG-1 genomic 
clone isolated from a genomic DNA library made from ES cells of a 129/Sy 
mouse, in order to maximize homology between the targeting plasmid and the 
target sequences. Homologous integration would create a deletion of 1356 
basepairs in the 5' end of the coding sequence of RAG-1. The resulting 
mutation would most likely be a null mutation, as the deletion encompasses 
about half of the coding sequence of RAG-1 and includes the putative 
nuclear localization signal and zinc-finger-like motif (Schatz et al., 
1989). Moreover, as the neo-selectable marker is introduced in the 
opposite transcriptional orientation, a polypeptide correctly initiated at 
the translation start site would probably be prematurely terminated at the 
level of the neo-gene. 
The targeting strategy was to isolate a number of G418-resistant clones and 
analyze them individually, since previous experience had shown that the 
negative selection step utilizing the HSV-thymidine kinase gene (Mansour 
et al., Nature 336, 348-352 (1988)) did not give a significant enrichment 
and that targeting frequencies among the G418-resistant clones are 
generally high enough for direct screening. 
Using AB1 embryonic stem cells (McMahon et al), targeted clones were 
obtained in two independent experiments, one (clone A103) out of 130 
G418-resistant clones in one experiment and one (clone G113) out of 117 
clones in another experiment. 
Generation of Chimeric Mice 
Litters from heterozygous mice were from birth housed in autoclaved 
micro-isolator cages and given autoclaved food and water. 
Chimaeras were produced as described in Bradley, 1987, the teachings of 
which are incorporated herein. Clone A103 was injected in C57BL/6 
blastocysts. From those implanted females that became pregnant 
(representing 194 implanted blastocysts), 29 male, 1 hermaphrodite and 15 
female chimaeras were born, with an average coat color chimerism of about 
75%. Of 27 chimeric males that were test mated to CD1 or C57BL/6 females, 
17 had live offspring and 12 were germline chimaeras. One out of eight 
female chimaeras had germline offspring. Clone A103 was injected in eight 
Balb/c blastocysts, which gave rise to two male and one female chimaeras. 
This female proved to be a germline chimeric. Clone G113 did not give rise 
to good chimaeras. 
Screening of Mice 
Mice were screened by Southern blot analysis on genomic DNA isolated 
according to Laird et al., 1991. The DNA samples were cut with Pst 1 or a 
combination of BamHI and Nco1 and the Southern blots were probed with 
probe B-X, a 1544 bp fragment containing RAG-1 coding sequences between 
positions 1837 and 3380 (Schatz et al., 1989). Southern blots were exposed 
using a Fujix Bio-Image Analyzer BAS2000. 
(129/Sv.times.CD1) F1 heterozygotes were intercrossed to produce 
homozygotes. Offspring was genotyped by Southern blot analysis of tail 
DNA. Data from one litter was analyzed by Southern Blot on tail DNA. Tail 
DNA was isolated from a litter of 13 mice of a (129/Sv.times.CD1) F1 
heterozygote intercross. DNA was cut with BamHI and Nco1, and hybridized 
to probe B-X. The upper band indicates the wild type allele, the lower 
band corresponds to the allele that has undergone homologous 
recombination. Three mice in the litter are homozygous, as they have only 
the lower band. The blot was stripped and hybridized with probe Bg1, which 
is complementary to sequences within the deletion generated by the 
targeting event. The three homozygous mice do not contain DNA sequences 
hybridizing to this probe. Of 20 newborn animals, 6 were homozygous, and 
33 out of 112 offspring genotyped after weaning were homozygous. 
Flow Cytometric Analysis 
10.sup.5 to 10.sup.6 cells were preincubated in 96 well round bottom dishes 
(Costar) for 20 minutes in 12.5 .mu.l containing 5 .mu.l of staining 
solution (composed of PBS/0.1% sodium azide/1% fetal calf serum), and 2.5 
.mu.l of each normal hamster, normal mouse (Jackson Immunology) and normal 
rat serum (Caltag). The preincubation with serum was omitted in the 
stainings with goat-antimouse. The samples were then stained by adding 
another 12.5 .mu.l of staining solution containing 0.25 .mu.l of 
antibodies, either biotinylated or conjugated with fluorescein 
isothiocyanate (FITC) or phycoerythrin (PE). The antibodies used were: 
2C11 for CD3-epsilon (Pharmingen), H57-597 for TCR alpha beta 
(Pharmingen), 53-6.7 for CD8 (Becton Dickinson), GK1.5 for CD4 
(Pharmingen), 53-2.1 for Thy1.2 (Pharmingen), PC 61 5.3 for 
1L2-receptor/CD25 (gift from Kunio Sano), goat-antimouse immunoglobulin 
(Caltag), RA3-6B2 for B220 (Boehringer Mannheim), 53-7.3 for CD5 (gift 
from Pablo Pereira), J11d (gift from Pablo Pereira), anti-Sca-1 (gift from 
Peggy Goodell), and M1/70 for Mac1 (Boehringer Mannheim). 
Cells were stained for 30 minutes, washed twice with staining solution, 
incubated in 25 .mu.l of staining solution containing streptavidin-biotin 
(Southern Biotechnology) at a 1:200 dilution, washed once in staining 
solution, once in PBS containing propidium-iodide and finally resuspended 
in 200 .mu.l of PBS. Cells were kept on ice during the staining procedure 
and spun in a refrigerated centrifuge. Flow cytometric analysis was 
carried out with a Becton Dickinson FACScan using FACScan software. Dead 
cells were gated out by means of propidium iodide staining. 5000 to 17000 
events were acquired using a large gate and the lymphoid population was 
analyzed with a narrower gate based upon forward scatter and side scatter. 
Southern and Northern Blot Analysis 
TCR .alpha. and .delta. rearrangements were analyzed by cutting genomic DNA 
from thymus with EcoRI and hybridizing with the 3'J.sub..delta.1 probe 
described in Chien et al., 1987 (gift from Yo-Ichi Shinkai). TCR .beta. 
rearrangements were analyzed by hybridizing with a 5'D.sub.1 probe, a 1.2 
kb Pst1 fragment isolated from cosmid 2.3 W7 (Malissen et al., 1984). The 
TCR .alpha. probe was a 0.6 kb BglII-BamHI fragment (Mombaerts et al., 
1991). Immunoglobulin heavy chain rearrangements were analyzed by cutting 
with EcoRI and hybridizing with a 1.9 kb BamHI-EcoRI J.sub.H probe (Sakano 
et al., 1980). 
The RAG-2 probe is a 246 bp fragment cloned into pUC12 with the following 
PCR primers: 5' ATGTCCCTGCAGATGGTAACA 3' (position 156 to 176 in Oettinger 
et al., 1990) and 5' GCCTTTGTATGAGCAAGTAGC 3' (position 401 to 381 in 
Oettinger et al., 1990). The lambda5 (Sakaguchi and Melchers, 1986) cDNA 
probe was a gift from Gene Oltz. 
Macroscopic Analysis 
Mice homozygous for the RAG-1 mutation are healthy and indistinguishable 
from their normal littermates by visual inspection, up to at least 21 
weeks of age, preferably maintained in a germ free environment. Two twelve 
old mutant males could readily fertilize either a homozygous or 
heterozygous mutant female. In the former case, twelve pups were born and 
in the latter, eleven pups. Both litters were raised successfully to 
weaning. 
The number of cells in the lymphoid organs of twelve mutant mice of varying 
ages (newborn and three to nine week old mice) were determined and cared 
with corresponding wild-type or heterozygous littermates. The thymus of 
the RAG-1 deficient mice contained 15 to 130 times less cells than the 
thymus of the wild-type or heterozygous littermates. One four week old 
mutant mouse had the highest number of thymocytes among all mutant mice 
examined but that was still 3.4.times.10.sup.6 compared to 
230.times.10.sup.6 thymocytes in a wild-type littermate. The numbers of 
non-erythroid cells in the spleen of mutant mice of three weeks or older 
were between five and nine times smaller than those in corresponding 
wild-type or heterozygous littermates. Visual inspection revealed a 
stroma-like structure in the site where the inguinal lymph nodes are 
located, but only a few non-erythroid cells, if any, could be recovered 
from these structures. No other obvious anatomical alterations were 
observed. 
For histology of the brain, mice were deeply anesthetized with avertin, 
perfused for 2 minutes with phosphate buffered saline and then with 4% 
paraformaldehyde in phosphate buffer (0.1M, pH 7.4) for 15 minutes. 
Following perfusion the brain was removed from the skull and stored in 
fresh paraformaldehyde overnight at 4.degree. C. The brains were bisected 
at the midline and embedded in polyester wax (polyethylene glycol 
distearate 400) according to the protocol of Mullen (1977). Serial ten 
micron sections were collected, mounted on gelatinized slides and stained 
with cresyl violet. 
As far as a potential neurological deficit is concerned, based on the 
report by Chun et al., (1990), no obvious clinical symptoms have been 
observed to date. The mutant mice are active, have a firm grip with their 
forelimbs, can hear sounds, can walk on a pencil, can sense heat and feel 
pain and can swim as well as their heterozygous or wild-type littermates. 
Absence of Mature B and T Lymphocytes 
Cells from thymus, spleen, lymph nodes, bone marrow and blood were 
subjected to flow cytometric analysis. The thymus isolated from four, 
five, or seven week old mice were analyzed by FACScan. The Forward scatter 
(FSC) horizontally, reflects the size of the cells; side scatter (SSC) 
vertically, corresponds to the granularity of the cells. The thymocytes of 
mutant mouse are shifted to higher scatter values. Analysis with CD3 
(FITC) horizontally and pan-TCR .alpha..beta. (PE) vertically demonstrates 
that no CD3 or TCR .alpha..beta. thymocytes exist in the mutant thymus. 
Analysis with CD8 (FITC) horizontally and CD4 (biotin-PE) vertically 
demonstrates that no CD8 CD4 double positive cells are observed in the 
mutant mouse thymus. Analysis with Thy 1.2F (FITC) horizontally and 
IL2-receptor/CD25 (biotin-PE) vertically demonstrates that most thymocytes 
in the mutant mouse express the IL2-receptor. 
The spleen and bone marrow were also analyzed by FACScan using samples from 
the same mice. A narrow gate was used on the splenocytes to visualize only 
the lymphoid cells, representing 72% of the non-erythroid cells in the 
wild-type mouse, and 32% in the mutant mouse. Analysis with IgM (FITC) 
horizontally and B220 (PE) vertically show that only B220 single positive 
cells exist in the mutant mouse. Even in the ungated population no 
B220-IgM double positive cells are found. Based on the control mouse, a 
narrow gate on the lymphoid population was used to examine bone marrow 
(21% of the total cells in the RAG-1 heterozygous mouse, and 13% of the 
total cells in the mutant mouse). Only dull B220 single positive cells are 
observed in the mutant mouse. 
The thymocytes of the RAG-1 deficient mouse are larger than those of 
wild-type or heterozygous mice, as indicated by the increased forward 
scatter. No CD3 positive or TCR .alpha..beta. positive cells have been 
observed. The thymocytes are CD8-CD4 double negative and almost all of 
them are IL2-receptor positive (FIG. 3D). Finally, they are Thy1 positive 
(FIG. 3d), bright J11d positive, bright Sca-1 positive and CD5 negative. 
Thus, thymocyte development seems to be arrested at an immature stage. 
The spleen does not contain any mature B cells as judged by the lack of 
staining by anti-IgM or anti-IgD antibody. About one third of the 
splenocytes (on a narrow gate for the lymphoid population) are B220 
positive and may represent an early stage of B cell differentiation. A 
small fraction of the splenocytes is Mac-1 positive and could be 
macrophages, natural killer cells, or neutrophils. Likewise, the bone 
marrow contains no mature, IgM or IgD positive cells. About one fourth of 
the bone marrow cells, with a narrow gate on the lymphoid population are 
B220 positive and could be early B-cell precursors. The intensity of the 
B220 staining is lower than the majority of the B220 positive cells in the 
bone marrow of the normal mouse. The composition of the lymphoid 
population in the lymphoid organs of the RAG-1 deficient mouse is similar 
to that of the scid mouse described by Bosma and Carrol, (1991). The serum 
IgM levels of seven mutant mice of five to sixteen weeks have also been 
measured by ELISA. IgM was non detectable. The serum IgM level is a 
sensitive indicator for leakiness in the case of the scid mouse. 
In summary, no mature B or T lymphocytes have been observed in the lymphoid 
organs of twelve RAG-1 deficient mice of up to the age of nine weeks and 
no serum IgM was detectable in mice up to the age of sixteen weeks. 
Establishment of Abelson-Transformed Lines 
The Abelson murine leukemia virus was used to transform bone marrow cells 
from a five week old female mutant mouse as well as from a wild-type 
female littermate, as described in Rosenberg and Baltimore, 1976. 
Transformation efficiency was determined by counting the number of 
colonies in soft agar per 10.sup.6 infected bone marrow cells. 
A similar number of cell lines grew up in both mice. Transformation 
efficiency was 1.19.times.10.sup.-4 for homozygote and 
8.74.times.10.sup.-5 for wild-type, as has been reported for scid mouse, 
Fulop, et al., Cell Immunol. 113, 192-201 (1988). All of the Abelson 
retrovirus-transformed lines are B220 positive and contain RAG-2 and 
lambda5 (Sakaguchi and Melchers, 1986) RNA (FIG. 5a and c). It is 
therefore likely that these cell lines originated from immature B cells. 
It is therefore likely that these cell lines originated from immature B 
cells. Mutant RAG-1 transcripts are present in all of the cell lines 
established from mutant mouse as determined by Northern blot analysis of 
expression of RAG-2, RAG-1 and lambda5 in bone marrow-derived 
Abelson-transformed lines. RNA was isolated from individual Abelson-lines. 
The blot was hybridized to (A) a RAG-2 probe (48 hr exposure); (B) the 
RAG-1 probe B-X (12 hr exposure); (C) a lambda5 cDNA probe (6 hr 
exposure); (D) a 1.5 kb AvaI-BamHI fragment from the human .beta.-actin 
gene. Thirteen Abelson-lines from RAG-1 deficient mouse were analyzed; 
three Abelson-lines from wild-type littermate were analyzed. The cell line 
represented by one Abelson-line from a wild-type littermate gave neither a 
RAG-1 nor a RAG-2 RNA band. This is not unusual because large variations 
in the levels of RAG-1 and RAG-2 RNA in Abelson-transformed lines from 
normal mice have been observed. 
In order to confirm that the absence of mature B and T lymphocytes is due 
to a defect in V(D)J recombination, Southern blot analysis was performed 
with DNA from the thymus and the Abelson-transformed lines. The 
3'J.sub..delta.1 probe described by Chien, et al., (1987), allows the 
analysis of rearrangements involving D.sub..delta.2 or J.sub..delta.1 and 
all V.sub..alpha. -J.sub..alpha. rearrangements (in the latter case the 
TCR.sub..delta. locus is deleted). DNA from 5.times.10.sup.5 cells was cut 
with EcoRI. The Southern blot was hybridized to the 3'J.delta.1 probe. The 
size of the germline fragment is 7.5 kb. AB1 embryonic stem cells; 
thymocytes from a four week old wild-type mouse; thymocytes from a 
homozygous mutant mouse that is a littermate of the wild-type mouse; 
thymocytes from a seven week old wild-type mouse; thymocytes from a 
homozygous mutant mouse that is a littermate of the wild-type mouse; 
thymocytes from a seven week old scid mouse were compared. There is no 
evidence for rearrangements in the thymus of the mutant mouse. The scid 
mouse has two faint bands which probably represent D-D or D-J 
rearrangements. The blot was stripped and hybridized to the 5'D.beta.1 
probe. No rearrangements are observed in the thymocytes from a homozygous 
mutant mouse that is a littermate of the wild-type mouse or the thymocytes 
from a homozygous mutant mouse that is a littermate of the wild-type 
mouse, and no signal is visible at the level of the germline band as the 
DNA sequences complementary to this probe are deleted (V-D-J 
rearrangement) or have an altered size (D-J rearrangement). A faint band 
is visible in the scid thymus and probably corresponds to a D-J 
rearrangement. The blot was stripped again and hybridized to a TCR 
C.sub..alpha. probe. The intensity of the bands serves to control for 
variations in amount of DNA loaded and Southern transfer efficiency. 
In summary, thymus DNA isolated from four and seven week old RAG-1 
deficient mice showed no rearrangements, whereas thymus DNA of a seven 
week old scid mouse showed a low level rearrangements which are presumably 
aberrant, Carroll and Bosman (1991). Using a 5' D.sub..beta.1 probe no 
rearrangements were observed at the TCR .beta. locus, whereas the scid 
thymus shows a faint additional band which probably represents a D-J 
rearrangement, as described Malissen, et al. (1984). A TCR C.sub..alpha. 
probe was used as a control for loading and Southern transfer efficiency 
of the DNA samples. 
The bone marrow-derived Abelson-transformed lines were similarly analyzed 
with an IgJ.sub.H probe. Southern blot analysis of endogenous IgH 
rearrangements in bone marrow-derived Abelson-transformed lines was 
performed. DNA was isolated from individual Abelson-lines and cut with 
EcoRI. The blot was hybridized with a 1.9 kb BamHI-EcoRI fragment 
containing J.sub.H3 and J.sub.H4, using the method of Sakano et al., 
Nature 286, 676-683 (1980). C57BL/6 liver; Abelson-lines derived from bone 
marrow of a five week old RAG-1 mutant mouse; and Abelson-lines derived 
from bone marrow of a wild-type littermate were compared. All of the cell 
lines revealed a germline configuration, but none of the lines derived 
from the wild-type mouse retained the IgH locus in a germline 
configuration. 
In conclusion, the inability to perform V(D)J recombination is the most 
likely explanation for the absence of mature B and T lymphocytes. 
Histological Analysis of the Brain 
In light of the reported presence of the RAG-1 transcript in the brain of 
the mouse (Chun et al., 1991), the brain was analyzed by histological 
means, although no obvious behavioral deficit was observed. Brains of 
seven and eight week old homozygous and wild-type littermates were 
analyzed with serial sagittal sections stained with cresyl violet. 
No significant defect could be found in the structure of the brains of the 
mutant mice when compared to control littermates. On gross examination all 
major external features of the RAG-1 deficient brain were normal, 
including its size and the relative placement of the various fissures and 
nerve roots. Histological exam revealed that, to a first approximation, 
most major nuclei found in the wild type littermate were present in the 
mutant mouse and were normal in size and cellular appearance. No 
structural alterations were observed. 
Particular attention was focused on the hippocampus and the cerebellum as 
these are the two regions that are believed to have the highest levels of 
RAG-1 expression by in situ hybridization, as reported by Chun, et al., 
(1991). Comparison of wild-type with mutant shows a nearly identical 
pattern of foliation of the cerebellar cortex. Similarly the 
macro-architecture of the hippocampus is unaltered in the mutant mouse. 
High magnification views of both cerebellum and hippocampus reveal no 
distortion of the micro-architecture of the mutant compared to the wild 
type brain. As these areas both are sensitive indicators of pattern 
alterations, as described by Nowakowsky (1984) and Joyner, et al., (1991), 
their normal appearance in the mutant suggest that the absence of RAG-1 
function does not interfere in a major way with developmental events such 
as neurogenesis, migration and differentiation. 
There was near identity in the foliation pattern of the cerebellar cortex 
and the close congruence of the structure of the hippocampi. Higher 
magnification of cerebellar cortex reveals no alternation in the density 
or Nissl morphology of cells in the molecular, Purkinje or granule cell 
layer between wild type and mutant. Likewise, the cytoarchitecture of the 
hippocampus is indistinguishable in wild-type and mutant brains 
(270.times.). 
RAG-1 and RAG-2 Are Both Required for V(D)J Recombination 
The generation and the initial analysis of mice with a mutation of the 
RAG-1 gene, which has been implicated in the regulation or the catalysis 
of V(D)J recombination (Schatz et al., 1980), show that this gene is 
necessary in vivo for V(D)J recombination to occur. The presence of RAG-2 
(Oettinger et al., 1990) transcripts in the Abelson-transformed lines 
derived from the RAG-1 mutant mouse indicates that the mutation introduced 
in the RAG-1 gene did not inhibit expression of the closely linked RAG-2 
gene. RAG-1 transcripts of a slightly larger size are observed in the 
RAG-1 deficient lines but are not likely to give rise to any functional 
protein. Taken together with the data on the RAG-2 deficient mice 
described by Shinkal et al., Cell (in press 1992), the results support the 
hypothesis that both the RAG-1 and RAG-2 gene products are required in 
vivo for V(D)J recombination. 
Immature B and T Cells 
Flow cytometric analysis of cells from lymphoid organs reveals the absence 
of mature B and T lymphocytes, which is most likely the result of the 
deficit in V(D)J recombination. B and T cell precursors need to produce 
functional antigen receptors on their surface in order to produce a T cell 
receptor do not undergo the process of positive selection. Thus, like in 
the scid mouse, the thymus of the RAG-1 mutant mouse remains small and 
contains immature, large, CD8 CD4 double negative thymocytes expressing 
the IL2- receptor. Likewise, the bone marrow and the spleen of the RAG-1 
mutant mouse contain a population of dull B220 positive cells, a fraction 
of which may represent pre-B cells. Some of these cells may serve as 
targets for transformation by the Abelson retrovirus. 
No Leakiness 
The combination of the flow cytometric data, Southern blot analysis and 
serum IgM ELISA measurements revealed no leakiness in the phenotype to 
date. 
The observations made to date are in contrast to those in the scid mouse. 
Using the 3'J.sub..delta.1 probe of Chien, et al., (1987), D-J 
rearrangements at the TCR .delta. locus have been reported for scid mice 
by Carroll and Bosma, (1991). As these mice grow older, some B and T cells 
express a functional antigen receptor and form an oligoclonal repertoire 
(Carroll and Bosma, 1988; Bosma, et al., 1988; Carroll, et al., 1989; 
Bosma and Carroll, 1991). As these mice grow older, some B and T cells 
express a functional antigen receptor and form an oligoclonal repertoire. 
This leakiness is partly the result of reversion of the mutation in 
individual lymphocyte progenitors. As a result, serum levels of IgM 
increase in scid mice as they age. 
Modifications and variations of the mutant animals, and methods of making 
and using them, will be obvious to those skilled in the art from the 
foregoing detailed description of the invention, and are intended to fall 
within the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mouse 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TTGCTATCTCTGTGGCATCGAGTGTTAACAACCAAGCTGCAGACATTCTAGCACTCTGGC60 
CGGGAGGCCTGTGGAGCAAGGTAGCTTAGCCAACATGGCTGCCTCCTTGCCGTCTACCCT120 
GAGCTTCAGTTCTGCACCCGATGAAATTCAACACCCACAAATCAAATTTTCCGAGTGGAA180 
ATTTAAGCTGTTTAGGGTGAGATCCTTTGAAAAGGCACCCGAAGAAGCACAGAAGGAGAA240 
GGATTCCTCAGAGGGGAAACCTTACCTAGAACAGTCTCCAGTAGTTCCAGAGAAGCCTGG300 
TGGTCAGAACTCAATTCTGACTCAACGAGCACTGAAACTCCATCCTAAATTTTCAAAGAA360 
ATTCCATGCTGATGGGAAGTCAAGCGACAAAGCAGTTCACCAAGCCAGGCTTAGACACTT420 
CTGCCGCATCTGTGGGAATCGTTTCAAGAGTGACGGGCACAGCCGGAGATACCCAGTCCA480 
CGGGCCCGTGGACGCTAAAACCCAAAGTCTTTTCCGAAAGAAGGAAAAAAGAGTCACTTC540 
CTGGCCAGACCTCATTGCCAGGATTTTCCGGATCGACGTGAAGGCAGATGTTGACTCCAT600 
CCACCCGACGGAATTCTGCCATGACTGTTGGAGCATCATGCACAGAAAGTTCAGCAGTTC660 
CCACAGTCAGGTCTACTTCCCAAGGAAAGTGACCGTGGAGTGGCACCCCCACACACCGTC720 
CTGTGACATCTGTTTTACTGCCCATCGGGGACTCAAGAGGAAGAGACATCAGCCCAATGT780 
GCAGCTCAGCAAGAAACTAAAAACTGTGCTCAACCACGCGAGACGGGACCGTCGCAAGAG840 
AACTCAGGCTAGGGTCAGCAGCAAGGAAGTCCTGAAGAAGATCTCCAACTGCAGTAAGAT900 
TCATCTCAGTACCAAGCTTCTTGCCGTGGACTTCCCAGCACACTTTGTGAAATCCATCTC960 
CTGCCAGATATGCGAACACATTCTGGCTGATCCCGTGGAGACCAGCTGCAAGCATCTATT1020 
CTGTAGGATCTGCATTCTCAGATGTCTCAAAGTCATGGGCAGCTATTGTCCCTCTTGCCG1080 
ATATCCGTGCTTCCCTACTGACCTGGAGAGCCCAGTGAAGTCCTTTCTGAACATCTTGAA1140 
TTCTCTCATGGTCAAGTGTCCCGCGCAAGATTGCAATGAGGAAGTGAGTCTGGAAAAATA1200 
TAACCACCATGTGTCAAGCCACAAAGAATCTAAAGAGACTTTGGTGCATATCAATAAAGG1260 
GGGACGGCCTCGCCAGCATCTCCTGTCACTGACGAGAAGGGCGCAGAAACATCGGCTGAG1320 
GGAGCTCAAGATTCAAGTCAAAGAATTTGCTGACAAAGAAGAAGGTGGAGATGTGAAGGC1380 
TGTCTGCTTGACATTGTTTCTCCTGGCACTGAGGGCGAGGAATGAGCACAGGCAAGCTGA1440 
TGAATTAGAGGCCATCATGCAAGGCAGGGGCTCCGGGCTTCAACCAGCTGTTTGCTTGGC1500 
CATCCGTGTCAATACCTTCCTCAGCTGTAGCCAATACCATAAGATGTACAGGACTGTGAA1560 
AGCTATCACTGGGAGGCAGATTTTTCAACCTTTGCATGCTCTTCGGAATGCCGAGAAAGT1620 
CCTTCTGCCAGGCTACCATCCCTTTGAGTGGCAGCCCCCACTGAAGAATGTGTCCTCCAG1680 
AACTGATGTTGGAATTATTGATGGGCTGTCTGGACTTGCCTCCTCTGTGGATGAGTACCC1740 
AGTAGATACCATTGCGAAGAGGTTCCGCTACGACTCTGCTTTGGTGTCTGCTTTGATGGA1800 
CATGGAAGAAGACATCTTGGAAGGCATGAGATCCCAAGATCTTGATGACTACCTGAATGG1860 
TCCCTTCACAGTGGTGGTAAAGGAGTCTTGCGATGGAATGGGGGATGTGAGTGAGAAGCT1920 
CGGGAGTGGGCCCGCAGTTCCAGAAAAGGCCGTTCGTTTCTCTTTCACAGTCATGAGAAT1980 
TACGATAGAGCATGGTTCACAGAACGTGAAGGTGTTTGAGGAACCCAAGCCCAATTCTGA2040 
ACTGTGTTGCAAGCCGTTGTGTCTTATGCTGGCAGATGAGTCTGACCATGAGACCCTTAC2100 
TGCTATTCTAAGCCCCCTCATTGCCGAGAGGGAGGCCATGAAGAGCAGTGAATTAACGCT2160 
GGAGATGGGAGGCATCCCCAGGACTTTTAAATTCATCTTCAGGGGCACTGGATACGATGA2220 
AAAACTTGTCCGGGAAGTAGAAGGCTTGGAAGCTTCTGGCTCAGTCTACATCTGTACACT2280 
CTGTGACACCACCCGTTTGGAAGCCTCTCAGAATCTTGTCTTCCAGTCCATAACCAGAAG2340 
CCACGCCGAGAACCTGCAGCGCTATGAGGTCTGGCGGTCCAATCCGTATCATGAGTCCGT2400 
GGAAGAGCTCCGGGACCGGGTGAAAGGGGTCTCTGCCAAACCTTTCATCGAGACAGTCCC2460 
TTCCATAGATGCGCTTCACTGTGACATTGGCAATGCAGCTGAATTCTATAAGATTTTCCA2520 
GCTGGAGATAGGGGAAGTGTATAAACATCCCAATGCCTCTAAAGAGGAAAGGAAGAGATG2580 
GCAGGCCACGCTGGACAAACATCTCCGGAAAAGGATGAACTTAAAACCAATCATGAGGAT2640 
GAATGGCAACTTTGCCCGGAAGCTTATGACCCAAGAGACTGTAGACGCAGTTTGTGAGTT2700 
AATTCCTTCTGAGGAGAGGCATGAAGCTCTCAGGGAGCTCATGGACCTTTACCTGAAGAT2760 
GAAACCCGTGTGGCGCTCTTCATGTCCCGCTAAAGAGTGTCCAGAGTCCCTCTGTCAGTA2820 
CAGTTTCAACTCACAGCGTTTCGCGGAACTCCTCTCCACCAAGTTCAAATATAGATACGA2880 
GGGCAAAATCACCAATTACTTTCACAAAACCTTGGCACATGTCCCTGAAATTATTGAAAG2940 
GGATGGCTCTATCGGGGCCTGGGCAAGTGAGGGAAATGAATCGGGTAACAAGCTGTTTAG3000 
ACGGTTTCGGAAAATGAATGCCAGGCAGTCCAAGTGCTATGAGATGGAAGATGTCCTGAA3060 
ACATCACTGGCTGTATACTTCAAAATACCTCCAGAAGTTTATGAATGCTCATAACGCGTT3120 
AAAAAGCTCTGGGTTTACCATGAACTCAAAGGAGACCTTAGGGGACCCTTTGGGCATTGA3180 
GGACTCTCTGGAAAGCCAAGATTCAATGGAGTTTTAAATAGGATCTCCACATAGAAGTTG3240 
GTATTTGCCAATGTGTTTTCCTTTGGGTTGCAGTGAGGTCTTCTCCTAGCACCTAGCACA3300 
TTGCCATGTGGGTGGGTCTTATCACCCAAGGGGTGACATGTT3342 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1040 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mouse 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetAlaAlaSerLeuProSerThrLeuSerPheSerSerProAlaAsp 
151015 
GluIleGlnHisProGlnIleLysPheSerGluTrpLysPheLysLeu 
202530 
PheArgValArgSerPheGluLysAlaProGluGluAlaGlnLysGlu 
354045 
LysAspSerSerGluGlyLysProTyrLeuGluGlnSerProValVal 
505560 
ProGluLysProGlyGlyGlnAsnSerIleLeuThrGlnArgAlaLeu 
65707580 
LysLeuHisProLysPheSerLysLysPheHisAlaAspGlyLysSer 
859095 
SerAspLysAlaValHisGlnAlaArgLeuArgHisPheCysArgIle 
100105110 
CysGlyAsnArgPheLysSerAspGlyHisSerArgArgTyrProVal 
115120125 
HisGlyProValAspAlaLysThrGlnSerLeuPheArgLysLysGlu 
130135140 
LysArgValThrSerTrpProAspLeuIleAlaArgIlePheArgIle 
145150155160 
AspValLysAlaAspValAspSerIleHisProThrGluPheCysHis 
165170175 
AspCysTrpSerIleMetHisArgLysPheSerSerSerHisSerGln 
180185190 
ValTyrPheProArgLysValThrValGluTrpHisProHisThrPro 
195200205 
SerCysAspIleCysPheThrAlaHisArgGlyLeuLysArgLysArg 
210215220 
HisGlnProHisValGlnLeuSerLysLysLeuLysThrValLeuAsn 
225230235240 
HisAlaArgArgAspArgArgLysArgThrGlnAlaArgValSerSer 
245250255 
LysGluValLeuLysLysIleSerAsnCysSerLysIleHisLeuSer 
260265270 
ThrLysLeuLeuAlaValAspPheProAlaHisPheValLysSerIle 
275280285 
SerCysGlnIleCysGluHisIleLeuAlaAspProValGluThrSer 
290295300 
CysLysHisLeuPheCysArgIleCysIleLeuArgCysLeuLysVal 
305310315320 
MetGlySerTyrCysProSerCysArgTyrProCysPheProThrAsp 
325330335 
LeuGluSerProValLysSerPheLeuAsnIleLeuAsnSerLeuAsn 
340345350 
ValLysCysProAlaGlnAspCysAsnGluGluValSerLeuGluLys 
355360365 
TyrAsnHisHisValSerSerHisLysGluSerLysGluThrLeuVal 
370375380 
HisIleAsnLysGlyGlyArgPheArgGlnHisLeuLeuSerLeuThr 
385390395400 
ArgArgAlaGlnLysHisArgLeuArgGluLeuLysIleGlnValLys 
405410415 
GluPheAlaAspLysGluGluGlyGlyAspValLysAlaValCysLeu 
420425430 
ThrLeuPheLeuLeuAlaLeuArgAlaArgAsnGluHisArgGlnAla 
435440445 
AspGluLeuGluAlaIleAsnGlnGlyArgGlySerGlyLeuGlnPro 
450455460 
AlaValCysLeuAlaIleArgValAsnThrPheLeuSerCysSerGln 
465470475480 
TyrHisLysMetTyrArgThrValLysAlaIleThrGlyArgGlnIle 
485490495 
PheGlnProLeuHisAlaLeuArgAsnAlaGluLysValLeuLeuPro 
500505510 
GlyTyrHisProPheGluTrpGlnProProLeuLysHisValSerSer 
515520525 
ArgThrAspValGlyIleIleAspGlyLeuSerGlyLeuAlaSerSer 
530535540 
ValAspGluTyrProValAspThrIleAlaLysArgPheArgTyrAsp 
545550555560 
SerAlaLeuValSerAlaLeuMetAspMetGluGluAspIleLeuGlu 
565570575 
GlyMetArgSerGlnAspLeuAspAspTyrLeuAsnGlyProPheThr 
580585590 
ValValValGluGluSerCysAspGlyMetGlyAspValSerGluLys 
595600605 
LeuGlySerGlyProAlaValProGluLysAlaValArgPheSerPhe 
610615620 
ThrValMetAlaIleThrIleGluHisGlySerGlnAsnValLysVal 
625630635640 
PheGluGluProLysProAsnSerGluLeuCysCysLysProLeuCys 
645650655 
LeuAsnLeuAlaAspGluSerAspHisGluThrLeuThrAlaIleLeu 
660665670 
SerProLeuIleAlaGluArgGluAlaMetLysSerSerGluLeuThr 
675680685 
LeuGluMetGlyGlyIleProAlaThrPheLysPheIlePheArgGly 
690695700 
ThrGlyTyrAspGluLysLeuValArgGluValGluGlyLeuGluAla 
705710715720 
SerGlySerValTyrIleCysThrLeuCysAspThrThrArgLeuGlu 
725730735 
AlaSerGlnAsnLeuValPheHisSerIleThrArgSerHisAlaGlu 
740745750 
AsnLeuGlnArgTyrGluValTrpArgSerAsnProTyrHisGluSer 
755760765 
ValGluGluLeuArgAspArgValLysGlyValSerAlaLysProPhe 
770775780 
IleGluThrValProSerIleAspAlaLeuHisCysAspIleGlyAsn 
785790795800 
AlaAlaGluPheTyrLysIlePheGlnLeuGluIleGlyGluValTyr 
805810815 
LysHisProAsnAlaSerLysGluGluArgLysArgTrpGlnAlaThr 
820825830 
LeuAspLysHisLeuArgLysArgMetAsnLeuLysProIleMetArg 
835840845 
MetAsnGlyAsnPheAlaArgLysLeuMetThrGlnGluThrValAsp 
850855860 
AlaValCysGluLeuIleProSerGluGluArgHisGluAlaLeuArg 
865870875880 
GluLeuMetAspLeuTyrLeuLysMetLysProValTrpArgSerSer 
885890895 
CysProAlaLysGluCysProGluSerLeuCysGlnTyrSerPheAsn 
900905910 
SerGlnArgPheAlaGluLeuLeuSerThrLysPheLysTyrArgTyr 
915920925 
GluGlyLysIleThrAsnTyrPheHisLysThrLeuAlaHisValPro 
930935940 
GluIleIleGluArgAspGlySerIleGlyAlaTrpAlaSerGluGly 
945950955960 
AsnGluSerGlyAsnLysLeuPheArgArgPheArgLysMetAsnAla 
965970975 
ArgGlnSerLysCysTyrGluMetGluAspValLeuLysHisHisTrp 
980985990 
LeuTyrThrSerLysTyrLeuGlnLysPheMetAsnAlaHisSerAla 
99510001005 
LeuLysSerSerGlyPheThrMetAsnSerLysGluThrLeuGlyAsp 
101010151020 
ProLeuGlyIleGluAspSerLeuGluSerGlnAspSerMetGluPhe 
1025103010351040 
__________________________________________________________________________