Transgenic non-human mammals
Patent No.: 4,736,866
Date of Patent: Apr 12, 1988
Leder, et al.
Philip (Chestnut Hill, MA), Stewart; Timothy A.
(San Francisco, CA)
President and Fellows of Harvard College
; 435/317.1; 435/6; 536/23.5; 800/18
435/6,172.3,240,317,320,240.1,240.2 935/70,76,59,111,32 800/1
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
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
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.
||Rous Sarcoma Virus (Chicken)
||Y73 Sarcoma Virus (Chicken)
||Fujinami (St Feline)
Sarcoma Virus (Chicken, Cat)
Marine Leukemia Virus (Mouse)
||Rochester-2 Sarcoma Virus (Chicken)
||Gardner-Rasheed Feline Sarcoma Virus (Cat)
Erythroblastosis Virus (Chicken)
||McDonough Feline Sarcoma Virus (Cat)
||Moloney Murine Sarcoma Virus (Mouse)
||3611 Murine Sarcoma.sup.+ Virus (Mouse)
Murine Sarcoma Virus (Rat) (Balb/c mouse; 2 loci)
Murine Sarcoma Virus (Rat)
||Kirsten Murine Sarcoma Virus (Rat)
||Avian MC29 Myelocytomatosis Virus (Chicken)
T10 Virus (Chicken)
|| Reticuloendotheliosis Virus (Turkey)
Sarcoma Virus (Woolly Monkey)
|| Avian Myelo
||FBJ Osteosarcoma Virus (Mouse)
|| Neuroblastomas (Human)
||Neuroblastoma, Leukemia Sarcoma Virus (Human)
|| Bursal Lymphomas
|| Mammary Carcionoma (Human)
|| Neuro, Glioblastoma (Rat)
|| Chicken AEV (Chicken)
|| Rasheed Sarcoma Virus (Rat)
||Carcinoma Virus MH2 (Chicken)
|| Myelocytomatosis OK10 (Chicken)
|| Avian myeloblastosis/ erythroblastosis Virus E26
|| 3611-MSV (Mouse)
|| 3611-MSV (Mouse)
|| Ki-MSV (Rat)
|| 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
DESCRIPTION OF THE PREFERRED
The drawings will first briefly be described.
FIG. 1 is a diagrammatic representation of a region of a plasmid
bearing the mouse myc gene and flanking regions.
FIG. 2 is a diagrammatic represenation of a region of a
plasmid, pA9, bearing the mouse mammary tumor virus long terminal
repeat (MMTV LTR) sequences.
FIGS. 3-6 and 8 are diagrammatic representations of activated oncogene
FIG. 7 is a diagrammatic representation of a probe useful for detecting
activated myc fusions.
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
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
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%
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
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
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.
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.
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.
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. 39749,
39745, 39747, 39748, and 39746.
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.
435/6,172.3,240,317,320,240.1,240.2 935/70,76,59,111,32 800/1
A transgenic non-human mammal all of whose germ cells and somatic cells
contain a recombinant activated oncogene sequence introduced into said
mammal, or an ancestor of said mammal,
at an embryonic stage.
2. The mammal of claim 1, a chromosome of said mammal
including an endogenous coding sequence substantially the same as a
coding sequence of said oncogene sequence.
3. The mammal of claim 2, said oncogene sequence being
integrated into a chromosome of said mammal at a site different from
the location of said endogenous coding sequence.
4. The mammal of claim 2 wherein transcription of said
oncogene sequence is under the control of a promoter sequence different
from the promoter sequence controlling the transcription of said
endogenous coding sequence.
5. The mammal of claim 4 wherein said promoter sequence controlling transcription of said oncogene sequence is inducible.
6. The mammal of claim 1 wherein said oncogene sequence comprises a coding sequence of a c-myc gene.
7. The mammal of claim 1 wherein transcription of said oncogene sequence is under the control of a viral promoter sequence.
8. The mammal of claim 7 wherein said viral promoter sequence comprises a sequence of an MMTV promoter.
9. The mammal of claim 7 wherein said viral promoter sequence comprises a sequence of an RSV promoter.
10. The mammal of claim 1 wherein transcription of said
oncogene sequence is under the control of a synthetic promoter
11. The mammal of claim 1, said mammal being a rodent.
12. The mammal of claim 11, said rodent being a mouse.
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Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Clark; Paul T.
- Today in Science History, event description for issue of this Oncomouse patent No. 4,736,866 on 12 Apr 1988.
- Today in Science History, event description for issue of first life-form patent No. 4,259,444 on 31 Mar 1981.
- Today in Science History, event description for date of appeal of first life-form patent rejection arguments before Supreme Court on 17 Mar 1980.
- Today in Science History, event description for date of decision on appeal of patent rejection by Supreme Court on 16 Jun 1980.