A transgenic non-human eukaryotic animal whose germ cells and somatic cells contain an activated oncogene sequence introduced into the animal, or an ancestor of the animal, at an embryonic stage.

BACKGROUND OF THE INVENTION 
This invention relates to transgenic animals. 
Transgenic animals carry a gene which has been introduced into the germline 
of the animal, or an ancestor of the animal, at an early (usually 
one-cell) developmental stage. Wagner et al. (1981) P.N.A.S. U.S.A. 78, 
5016; and Stewart et al. (1982) Science 217, 1046 describe transgenic mice 
containing human globin genes. Constantini et al. (1981) Nature 294, 92; 
and Lacy et al. (1983) Cell 34, 343 describe transgenic mice containing 
rabbit globin genes. McKnight et al. (1983) Cell 34, 335 describes 
transgenic mice containing the chicken transferrin gene. Brinster et al. 
(1983) Nature 306, 332 describes transgenic mice containing a functionally 
rearranged immunoglobulin gene. Palmiter et al. (1982) Nature 300, 611 
describes transgenic mice containing the rat growth hormone gene fused to 
a heavy metal-inducible metalothionein promoter sequence. Palmiter et al. 
(1982) Cell 29, 701 describes transgenic mice containing a thymidine 
kinase gene fused to a metalothionein promoter sequence. Palmiter et al. 
(1983) Science 222, 809 describes transgenic mice containing the human 
growth hormone gene fused to a metalothionein promoter sequence. 
SUMMARY OF THE INVENTION 
In general, the invention features a transgenic non-human eukaryotic animal 
(preferably a rodent such as a mouse) whose germ cells and somatic cells 
contain an activated oncogene sequence introduced into the animal, or an 
ancestor of the animal, at an embryonic stage (preferably the one-cell, or 
fertilized oocyte, stage, and generally not later than about the 8-cell 
stage). An activated oncogene sequence, as the term is used herein, means 
an oncogene which, when incorporated into the genome of the animal, 
increases the probability of the development of neoplasms (particularly 
malignant tumors) in the animal. There are several means by which an 
oncogene can be introduced into an animal embryo so as to be chromosomally 
incorporated in an activated state. One method is to transfect the embryo 
with the gene as it occurs naturally, and select transgenic animals in 
which the gene has integrated into the chromosome at a locus which results 
in activation. Other activation methods involve modifying the oncogene or 
its control sequences prior to introduction into the embryo. One such 
method is to transfect the embryo using a vector containing an already 
translocated oncogene. Other methods are to use an oncogene whose 
transcription is under the control of a synthetic or viral activating 
promoter, or to use an oncogene activated by one or more base pair 
substitutions, deletions, or additions. 
In a preferred embodiment, the chromosome of the transgenic animal includes 
an endogenous coding sequence (most preferably the c-myc gene, hereinafter 
the myc gene), which is substantially the same as the oncogene sequence, 
and transcription of the oncogene sequence is under the control of a 
promoter sequence different from the promoter sequence controlling 
transcription of the endogenous coding sequence. The oncogene sequence can 
also be under the control of a synthetic promoter sequence. Preferably, 
the promoter sequence controlling transcription of the oncogene sequence 
is inducible. 
Introduction of the oncogene sequence at the fertilized oocyte stage 
ensures that the oncogene sequence will be present in all of the germ 
cells and somatic cells of the transgenic animal. The presence of the 
onocogene sequence in the germ cells of the transgenic "founder" animal in 
turn means that all of the founder animal's descendants will carry the 
activated oncogene sequence in all of their germ cells and somatic cells. 
Introduction of the oncogene sequence at a later embryonic stage might 
result in the oncogene's absence from some somatic cells of the founder 
animal, but the descendants of such an animal that inherit the gene will 
carry the activated oncogene in all of their germ cells and somatic cells. 
Any oncogene or effective sequence thereof can be used to produce the 
transgenic mice of the invention. Table 1, below, lists some known viral 
and cellular oncogenes, many of which are homologous to DNA sequences 
endogenous to mice and/or humans, as indicated. The term "oncogene" 
encompasses both the viral sequences and the homologous endogenous 
sequences. 
TABLE 1 
______________________________________ 
Abbreviation Virus 
______________________________________ 
src Rous Sarcoma Virus 
(Chicken) 
yes Y73 Sarcoma Virus 
(Chicken) 
fps Fujinami (St Feline) 
Sarcoma Virus 
(Chicken, Cat) 
abl Abelson Marine 
Leukemia Virus 
(Mouse) 
ros Rochester-2 Sarcoma 
Virus (Chicken) 
fgr Gardner-Rasheed 
Feline Sarcoma 
Virus (Cat) 
erbB Avian Erythroblastosis 
Virus (Chicken) 
fms McDonough Feline 
Sarcoma Virus (Cat) 
mos Moloney Murine 
Sarcoma Virus (Mouse) 
raf 3611 Murine Sarcoma.sup.+ 
Virus (Mouse) 
Ha-ras-1 Harvey Murine 
Sarcoma Virus (Rat) 
(Balb/c mouse; 2 loci) 
Ki-ras 2 Kirsten Murine Sarcoma 
Virus (Rat) 
Ki-ras 1 Kirsten Murine Sarcoma 
Virus (Rat) 
myc Avian MC29 
Myelocytomatosis Virus 
(Chicken) 
myt Avian Myelo 
Blastomas (Chicken) 
fos FBJ Osteosarcoma 
Virus (Mouse) 
ski Avian SKV T10 Virus 
(Chicken) 
rel Reticuloendotheliosis 
Virus (Turkey) 
sis Simian Sarcoma Virus 
(Woolly Monkey) 
N-myc Neuroblastomas (Human) 
N-ras Neuroblastoma, Leukemia 
Sarcoma Virus (Human) 
Blym Bursal Lymphomas 
(Chicken) 
mam Mammary Carcionoma 
(Human) 
neu Neuro, Glioblastoma 
(Rat) 
ertAl Chicken AEV (Chicken) 
ra-ras Rasheed Sarcoma Virus 
(Rat) 
mnt-myc Carcinoma Virus MH2 
(Chicken) 
myc Myelocytomatosis OK10 
(Chicken) 
myb-ets Avian myeloblastosis/ 
erythroblastosis Virus 
E26 (Chicken) 
raf-2 3611-MSV (Mouse) 
raf-1 3611-MSV (Mouse) 
Ha-ras-2 Ki-MSV (Rat) 
erbB Erythroblastosis virus 
(Chicken) 
______________________________________ 
The animals of the invention can be used to test a material suspected of 
being a carcinogen, by exposing the animal to the material and determining 
neoplastic growth as an indicator of carcinogenicity. This test can be 
extremely sensitive because of the propensity of the transgenic animals to 
develop tumors. This sensitivity will permit suspect materials to be 
tested in much smaller amounts than the amounts used in current animal 
carcinogenicity studies, and thus will minimize one source of criticism of 
current methods, that their validity is questionable because the amounts 
of the tested material used is greatly in excess of amounts to which 
humans are likley to be exposed. Furthermore, the animals will be expected 
to develop tumors much sooner because they already contain an activated 
oncogene. The animals are also preferable, as a test system, to bacteria 
(used, e.g., in the Ames test) because they, like humans, are vertebrates, 
and because carcinogenicity, rather than mutogenicity, is measured. 
The animals of the invention can also be used as tester animals for 
materials, e.g. antioxidants such as beta-carotine or Vitamin E, thought 
to confer protection against the development of neoplasms. An animal is 
treated with the material, and a reduced incidence of neoplasm 
development, compared to untreated animals, is detected as an indication 
of protection. The method can further include exposing treated and 
untreated animals to a carcinogen prior to, after, or simultaneously with 
treatment with the protective material. 
The animals of the invention can also be used as a source of cells for cell 
culture. Cells from the animals may advantageously exhibit desirable 
properties of both normal and transformed cultured cells; i.e., they will 
be normal or nearly normal morphologically and physiologically, but can, 
like cells such as NIH 3T3 cells, be cultured for long, and perhaps 
indefinite, periods of time. Further, where the promoter sequence 
controlling transcription of the oncogene sequence is inducible, cell 
growth rate and other culture characteristics can be controlled by adding 
or eliminating the inducing factor. 
Other features and advantages of the invention will be apparent from the 
description of the preferred embodiments, and from the claims.

MMTV-MYC FUSED GENES 
Gene fusions were made using the mouse myc gene and the MMTV LTR. The myc 
gene is known to be an activatable oncogene. (For example, Leder et al. 
(1983) Science 222, 765 explains how chromosomal translocations that 
characterize Burkitt's Lymphoma and mouse plasmacytomas result in a 
juxtaposition of the myc gene and one of the immunoglobulin constant 
regions; amplification of the myc gene has also been observed in 
transformed cell lines.) FIG. 1 illustrates the subclone of the mouse myc 
gene which provided the myc regions. 
The required MMTV functions were provided by the pA9 plasmid (FIG. 2) that 
demonstrated hormone inducibility of the p21 protein; this plasmid is 
described in Huang et al. (1981) Cell 27, 245. The MMTV functions on pA9 
include the region required for glucocorticoid control, the MMTV promoter, 
and the cap site. 
The above plasmids were used to construct the four fusion gene contructions 
illustrated in FIGS. 3-6. The constructions were made by deleting from pA9 
the Sma-EcoRI region that included the P21 protein coding sequences, and 
replacing it with the four myc regions shown in the Figures. Procedures 
were the conventional techniques described in Maniatis et al. (1982) 
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory). 
The restriction sites shown in FIG. 1 are Stul (St), Smal (Sm), EcoRI (R), 
HindIII (H), Pvul (P), BamHl (B), Xbal (X), and ClaI (C). The solid arrows 
below the constructions represent the promoter in the MMTV LTR and in the 
myc gene. The size (in Kb) of the major fragment, produced by digestion 
with BamHI and ClaI, that will hybridize to the myc probe, is shown for 
each construction. 
MMTV-H3 myc (FIG. 5) was constructed in two steps: Firstly, the 4.7 Kb Hind 
III myc fragment which contains most of the myc sequences was made blunt 
with Klenow polymerase and ligated to the pA9 Smal-EcoRI vector that had 
been similarly treated. This construction is missing the normal 3' end of 
the myc gene. In order to introduce the 3' end of the myc gene, the 
Pvul-Pvul fragment extending from the middle of the first myc intron to 
the pBR322 Pvul site in the truncated MMTV-H3 myc was replaced by the 
related Pvul-Pvul fragment from the mouse myc subclone. 
The MMTV-Xba myc construction (FIG. 3) was produced by first digesting the 
MMTV-Sma myc plasmid with Smal and Xbal. The Xbal end was then made blunt 
with Klenow polymerase and the linear molecule recircularized with T4 DNA 
ligase. The MMTV-Stu myc (FIG. 6) and the MMTV-Sma myc (FIG. 4) 
constructions were formed by replacing the P21 protein coding sequences 
with, respectively, the Stul-EcoRI or Smal-EcoRI myc fragments (the EcoRI 
site is within the pBR322 sequences of the myc subclone). As shown in FIG. 
1, there is only one Stul site within the myc gene. As there is more than 
one Smal site within the myc gene (FIG. 4), a partial Smal digestion was 
carried out to generate a number of MMTV-Sma myc plasmids; the plasm1d 
illustrated in FIG. 4 was selected as not showing rearrangements and also 
including a sufficiently long region 5' of the myc promoter (approximately 
1 Kb) to include myc proximal controlling regions. 
The constructions of FIGS. 4 and 6 contain the two promoters naturally 
preceding the unactivated myc gene. The contruction of FIG. 5 has lost 
both myc promoters but retains the cap site of the shorter transcript. The 
construction of FIG. 3 does not include the first myc exon but does 
include the entire protein coding sequence. The 3' end of the myc sequence 
in all of the illustrated constructions is located at the HindIII site 
approximately 1 kb 3' to the myc polyA addition site. 
These constructions were all checked by multiple restriction enzyme 
digestions and were free of detectable rearrangements. 
PRODUCTION OF TRANSGENIC MICE CONTAINING MMTV-MYC FUSIONS 
The above MMTV-myc plasmids were digested with SalI and EcoRI (each of 
which cleaves once within the pBR322 sequence) and separately injected 
into the male pronuclei of fertilized one-cell mouse eggs; this resulted 
in about 500 copies of linearized plasmid per pronucleus. The injected 
eggs were then transferred to pseudo-pregnant foster females as described 
in Wagner et al. (1981) P.N.A.S. U.S.A. 78, 5016. The eggs were derived 
from a CD-1 X C57Bl/6J mating. Mice were obtained from the Charles River 
Laboratories (CD.sup.R -1-Ha/Icr (CD-1), an albino outbred mouse) and 
Jackson Laboratories (C57Bl/6J), and were housed in an environmentally 
controlled facility maintained on a 10 hour dark: 14 hour light cycle. The 
eggs in the foster females were allowed to develop to term. 
ANALYSIS OF TRANSGENIC MICE 
At four weeks of age, each pup born was analyzed using DNA taken from the 
tail in a Southern hybridization, using a .sup.32 P DNA probe (labeled by 
nick-translation). In each case, DNA from the tail was digested with BamHI 
and ClaI and probed with the .sup.32 P-labeled BamHI/HindIII probe from 
the normal myc gene (FIG. 1). 
The DNA for analysis was extracted from 0.1-1.5 cm sections of tail, by the 
method described in Davis et al. (1980) in Methods in Enzymology, Grossman 
et al., eds., 65, 404, except that one chloroform extraction was performed 
prior to ethanol precipitation. The resulting nucleic acid pellet was 
washed once in 80% ethanol, dried, and resuspended in 300 .mu.l of 1.0 mM 
Tris, pH 7.4, 0.1 mM EDTA. 
Ten .mu.l of the tail DNA preparation (approximately 10 .mu.g DNA) were 
digested to completion, electrophoresed through 0.8% agarose gels, and 
transferred to nitrocellulose, as described in Southern (1975) J. Mol. 
Biol. 98, 503. Filters were hybridized overnight to probes in the presence 
of 10% dextran sulfate and washed twice in 2 X SSC, 0.1% SDS at room 
temperature and four times in 0.1 X SSC, 0.1% SDS at 64.degree. C. 
The Southern hybridizations indicated that ten founder mice had retained an 
injected MMTV-myc fusion. Two founder animals had integrated the myc gene 
at two different loci, yielding two genetically distinct lines of 
transgenic mice. Another mouse yielded two polymorphic forms of the 
integrated myc gene and thus yielded two genetically distinct offspring, 
each of which carried a different polymorphic form of the gene. Thus, the 
10 founder animals yielded 13 lines of transgenic offspring. 
The founder animals were mated to uninjected animals and DNA of the 
resulting thirteen lines of transgenic offspring analyzed; this analysis 
indicated that in every case the injected genes were transmitted through 
the germline. Eleven of the thirteen lines also expressed the newly 
acquired MMTV-myc genes in at least one somatic tissue; the tissue in 
which expression was most prevelant was salivary gland. 
Transcription of the newly acquired genes in tissues was determined by 
extracting RNA from the tissues and assaying the RNA in an Sl nuclease 
protection procedure, as follows. The excised tissue was rinsed in 5.0 ml 
cold Hank's buffered saline and total RNA was isolated by the method of 
Chrigwin et al. (1979) Biochemistry 18, 5294, using the CsCl gradient 
modification. RNA pellets were washed twice by reprecipitation in ethanol 
and quantitated by absorbance at 260 nm. An appropriate single stranded, 
uniformly labeled DNA probe was prepared as described by Ley et al. (1982) 
PNAS USA 79, 4775. To test for transcription of the MMTV-Stu myc fusion of 
FIG. 6, for example, the probe illustrated in FIG. 7 was used. This probe 
extends from a Smal site 5' to the first myc exon to an Sstl site at the 
3' end of the first myc exon. Transcription from the endogenous myc 
promoters will produce RNA that will protect fragments of the probe 353 
and 520 base pairs long; transcription from the MMTV promoter will 
completely protect the probe and be revealed as a band 942 base pairs 
long, in the following hybridization procedure. 
Labelled, single-stranded probe fragments were isolated on 8M urea 5% 
acrylamide gels, electroeluted, and hybridized to total RNA in a 
modification of the procedure of Berk et al. (1977) Cell 12, 721. The 
hybridization mixture contained 50,000 cpm to 100,000 cpm of probe 
(SA=10.sup.8 cpm/.mu.g), 10 .mu.g total cellular RNA, 75% formamide, 500 
mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, as described in Battey et al. 
(1983) Cell 34, 779. Hybridization temperatures were varied according to 
the GC content in the region of the probe expected to hybridize to mRNA. 
The hybridizations were terminated by the addition of 1500 units of Sl 
nuclease (Boehringer Mannheim). Sl nuclease digestions were carried out at 
37.degree. C. for 1 hour. The samples were then ethanol-precipitated and 
electrophoresed on thin 8M urea 5% acrylamide gels. 
Northern hybridization analysis was also carried out, as follows. Total RNA 
was electrophoresed through 1% formaldehyde 0.8% agarose gels, blotted to 
nitrocellulose filters (Lehrach et al. (1979) Biochemistry 16, 4743), and 
hybridized to nick-translated probes as described in Taub et al. (1982) 
PNAS USA 79, 7837. The tissues analyzed were thymus, pancreas, spleen, 
kidney, testes, liver, heart, lung, skeletal muscle, brain, salivary 
gland, and preputial gland. 
Both lines of mice which had integrated and were transmitting to the next 
generation the MMTV-Stu myc fusion (FIG. 6) exhibited transcription of the 
fusion in salivary gland, but in no other tissue. 
One of two lines of mice found to carry the MMTV-Sma myc fusion (FIG. 4) 
expressed the gene fusion in all tissues examined, with the level of 
expression being particularly high in salivary gland. The other line 
expressed the gene fusion only in salivary gland, spleen, testes, lung, 
brain, and preputial gland. 
Four lines of mice carried the MMTV-H3 myc fusion (FIG. 5). In one, the 
fusion was transcribed in testes, lung, salivary gland, and brain; in a 
second, the fusion was transcribed only in salivary gland; in a third, the 
fusion was transcribed in none of the somatic tissues tested; and in a 
fourth, the fusion was transcribed in salivary gland and intestinal 
tissue. 
In two mouses lines found to carry the MMTV-Xba myc fusion, the fusion was 
transcribed in testes and salivary gland. 
RSV-MYC FUSED GENES 
Referring to FIG. 8, the plasmid designated RSV-S107 was generated by 
inserting the EcoRI fragment of the S107 plasmacytoma myc gene, (Kirsch et 
al. (1981) Nature 293, 585) into a derivative of the Rous Sarcoma Virus 
(RSV) enhancer-containing plasmid (pRSVcat) described in Gorman et al. 
(1982) PNAS USA 79, 6777, at the EcoRI site 3' to the RSV enhancer 
sequence, using standard recombinant DNA techniques. All chloramphenicol 
acetyl transferase and SV40 sequences are replaced in this vector by the 
myc gene; the RSV promoter sequence is deleted when the EcoRl fragments 
are replaced, leaving the RSV enhancer otherwise intact. The original 
translocation of the myc gene in the S107 plasmacytoma deleted the two 
normal myc promoters as well as a major portion of the untranslated first 
myc exon, and juxtaposed, 5' to 5', the truncated myc gene next to the 
.alpha. immunoglobulin heavy chain switch sequence. 
The illustrated (FIG. 8) regions of plasmid RSV-S107 are: crosshatched, RSV 
sequences; fine-hatched, alpha 1 coding sequences; left-hatched, 
immunoglobulin alpha switch sequences; right-hatched, myc exons. The thin 
lines flanking the RSV-S107 myc exon represent pBR322 sequences. The 
marked restriction enzyme sites are: R, EcoRI; X, Xbal; P, Pst 1; K, Kpn 
1; H, HindIII; B, BamHI. The sequences used for three probes used in 
assays described herein (C-.alpha., .alpha.-sw and c-myc) are marked. 
PRODUCTION OF TRANSGENIC MICE 
Approximately 500 copies of the RSV-S107 myc plasmid (linearized at the 
unique Kpn-1 site 3' to the myc gene) were injected into the male 
pronucleus of eggs derived from a C57BL/6J x CD-1 mating. Mice were 
obtained from Charles River Laboratories (CD-1, an albino outbred mouse) 
and from Jackson Laboratories (C57BL/6J). These injected eggs were 
transferred into pseudopregnant foster females, allowed to develop to 
term, and at four weeks of age the animals born were tested for retention 
of the injected sequences by Southern blot analysis of DNA extracted from 
the tail, as described above. Of 28 mice analyzed, two males were found to 
have retained the new genes and both subsequently transmitted these 
sequences through the germline in a ratio consistent with Mendelian 
inheritance of single locus. 
First generation transgenic offspring of each of these founder males were 
analyzed for expression of the rearranged myc genes by assaying RNA 
extracted from the major internal tissues and organs in an Sl nuclease 
protection assay, as described above. The hearts of the offspring of one 
line showed aberrant myc expression; the other 13 tissues did not. 
Backcrossing (to C57Bl/6J) and in-breeding matings produced some transgenic 
mice which did not demonstrate the same restriction site patterns on 
Southern blot analysis as either their transgenic siblings or their 
parents. In the first generation progeny derived from a mating between the 
founder male and C57BL/6J females, 34 F1 animals were analyzed and of 
these, 19 inherited the newly introduced gene, a result consistent with 
the founder being a heterozygote at one locus. However, of the 19 
transgenic mice analyzed, there were three qualitatively different 
patterns with respect to the more minor myc hybridizing fragments. 
In order to test the possibility that these heterogenous genotypes arose as 
a consequence of multiple insertions and/or germline mosacism in the 
founder, two F1 mice (one carrying the 7.8 and 12 Kb BamHI bands, and the 
other carrying only the 7.8 Kb BamHI band) were mated and the F2 animals 
analyzed. One male born to the mating of these two appeared to have 
sufficient copies of the RSV-S107 myc gene to be considered as a candidate 
for having inherited the two alleles; this male was backcrossed with a 
wild-type female. All 23 of 23 backcross offspring analyzed inherited the 
RSV-S107 myc genes, strongly suggesting that the F2 male mouse had 
inherited two alleles at one locus. Further, as expected, the high 
molecular weight fragment (12 Kb) segregated as a single allele. 
To determine whether, in addition to the polymorphisms arising at the DNA 
level, the level of aberrant myc expression was also altered, heart mRNA 
was analyzed in eight animals derived from the mating of the above double 
heterozygote to a wild-type female. All eight exhibited elevated myc mRNA, 
with the amount appearing to vary between animals; the lower levels of 
expression segregated with the presence of the 12 Kb myc hybridizing band. 
The level of myc mRNA in the hearts of transgenic mice in a second 
backcross generation also varied. An F1 female was backcrossed to a 
C57Bl/6J male to produce a litter of seven pups, six of which inherited 
the RSV-S107 myc genes. All seven of these mice were analyzed for 
expression. Three of the six transgenic mice had elevated levels of myc 
mRNA in the hearts whereas in the other three the level of myc mRNA in the 
hearts was indistinguishable from the one mouse that did not carry the 
RSV-S107 myc gene. This result suggests that in addition to the one 
polymorphic RSV-S107 myc locus from which high levels of heart-restricted 
myc mRNA were transcribed, there may have been another segregating 
RSV-S107 myc locus that was transcriptionally silent. 
CARCINOGENICITY TESTING 
The animals of the invention can be used to test a material suspected of 
being a carcinogen, as follows. If the animals are to be used to test 
materials thought to be only weakly carcinogenic, the transgenic mice most 
susceptible of developing tumors are selected, by exposing the mice to a 
low dosage of a known carcinogen and selecting those which first develop 
tumors. The selected animals and their descendants are used as test 
animals by exposing them to the material suspected of being a carcinogen 
and determining neoplastic growth as an indicator of carcinogenicity. Less 
sensitive animals are used to test more strongly carcinogenic materials. 
Animals of the desired sensitivity can be selected by varying the type and 
concentration of known carcinogen used in the selection process. When 
extreme sensitivity is desired, the selected test mice can consist of 
those which spontaneously develop tumors. 
TESTING FOR CANCER PROTECTION 
The animals of the invention can be used to test materials for the ability 
to confer protection against the development of neoplasms. An animal is 
treated with the material, in parallel with an untreated control 
transgenic animal. A comparatively lower incidence of neoplasm development 
in the treated animal is detected as an indication of protection. 
TISSUE CULTURE 
The transgenic animals of the invention can be used as a source of cells 
for cell culture. Tissues of transgenic mice are analyzed for the presence 
of the activated oncogene, either by directly analyzing DNA or RNA, or by 
assaying the tissue for the protein expressed by the gene. Cells of 
tissues carrying the gene can be cultured, using standard tissue culture 
techniques, and used, e.g., to study the functioning of cells from 
normally difficult to culture tissues such as heart tissue. 
DEPOSITS 
Plasmids bearing the fusion genes shown in FIGS. 3, 4, 5, 6, and 8 have 
been deposited in the American Type Culture Collection, Rockville, Md., 
and given, respectively, ATCC Accession Nos. 39745, 39746, 39747, 39748, 
and 39749. 
OTHER EMBODIMENTS 
Other embodiments are within the following claims. For example, any species 
of transgenic animal can be employed. In some circumstances, for instance, 
it may be desirable to use a species, e.g., a primate such as the rhesus 
monkey, which is evolutionarily closer to humans than mice.