Transgenic
animals
GLENN
in biomedical
research
T. MERLINO
Laboratory Maryland
of Molecular 20892, USA
Biology,
National
Cancer
Institute,
The advent of transgenic technology, in which foreign genetic information is stably introduced into the mammalian germ line, has dramatically enhanced our basic knowledge of physiologic and pathologic processes. Transgenic animals created by these genetic manipulations are being used to provide insights into gene regulation, development, pathogenesis, and the treatment of disease. Furthermore, transgenic biotechnology holds great promise for the creation of genetically superior livestock and the industrial production of precious pharmaceuticals. It is evident now that the study and use of transgenic animals will significantly improve the human condition. Merlino, G. T. Transgenic animals in biomedical research. FASEB J. 5: 2996-3001; 1991. ABSTRACT
Key Words: biotechnology
.
gene regulation . gene therapy targeted gene inactivation
.
cancer
.
mutation
ABILITY TO PRECISELY AND PERMANENTLY modify genetic information by introducing foreign DNA into the germ line of animals has elevated the potential of biological research to an unprecedented level. This review describes how the study of transgenic animals has contributed to our ability to understand gene regulation, development, and the molecular basis of disease, and has facilitated improvements in gene therapy and biotechnology. This field has been explosive in its growth, and hundreds of reports are published yearly describing the generation, characterization, and use of transgenic animals. It is beyond the scope of this review to recount all this work, and therefore I have not discussed or cited many important contributions, as well as entire fields such as immunology; however, several excellent and comprehensive reviews of specialized areas are available and can be consulted (1-6). Instead, I will attempt to instill an understanding and appreciation of basic principles, describe recent developments, and speculate on future trends. Three main methods have been used to transfer genes into
THE
the
mouse
germ
line.
The
most
efficient
and
commonly
used
technique is the direct microinjection of DNA fragments into the pronuclei of one-cell embryos, which are then returned to the oviduct of a foster mother to complete development (Fig. 1A). In a less arduous method, cleavage-stage embryos are exposed to recombinant retroviruses in vitro and are returned to the uterus of a foster mother (Fig. 1B). Finally, recent technical advancements have permitted the in vitro maintenance, genetic manipulation, and selection of pleuripotent embryonic stem (ES)1 cells derived from the inner cell mass (Fig. 1C). Upon reintroduction into the embryonic blastocoel, ES cells that have been subjected to gene manipulation
can
colonize
the
line (7, 8). It is useful, genetic manipulations
embryo
and
contribute
to the
Institutes
of Health,
Bethesda,
tations
are often inactivation of an eign DNA (Fig. method available
recessive and are usually the results of the endogenous gene by the integration of for2D). Gene targeting is the most efficient to produce loss-of-function mutations.
REGULATION
OF
GENE
EXPRESSION
Gene regulation has been examined extensively in vitro by expressing foreign gene constructs in cultured cells. However, the relevance of many cell culture studies to actual in vivo events is tenuous. Transgenic animals provide a true in vivo environment for evaluating the mechanisms by which gene expression is modulated in the adult and during development. Detailed analysis of myriad gene regulatory elements (promoters and enhancers) in the context of the whole animal has led to remarkable discoveries about gene function, and also to the development of more sophisticated tools for targeting the expression of heterologous genes to specific types of cells (2, 4). For example, regulatory elements that control the complex tissue- and stage-specific pattern of a-fetoprotein expression during mouse embryogenesis have been identified by reproducing their activity in transgenic mice (9). Transgenic mice have also been used to identify regulatory control elements that are localized a great distance from the coding region of the gene. In the human 13-globin locus distant control elements, called locus control regions (LCR) or locus activation regions, are responsible for maximum appropriate expression in transgenic mice (see ref 10 for review). In the absence of specific DNase I hypersensitive sites in the LCR region, /3-globin expression is reduced approximately 300-fold in transgenic mice. These regions may serve as nuclear matrix attachment sites, as binding sites for topoisomerase II or as erythroid-specific enhancers of replication (10). A serious problem in achieving efficient and predictable transgene expression is the potentially deleterious effect of endogenous genomic sequences near the site of integration. The presence of LCRs within transgenes may overcome the influence of these flanking sequences. Specific regions of mammalian DNA, A-elements, have been identified which mediate attachment of the chromatin to the nuclear scaffold (11). Reporter genes flanked by A-elements exhibit elevated expression that is insertion site-independent in stably transfected cultured cells. The availability of control elements that would render a transgene free from the unpredictable influences of the mouse genome would enable copy numberdependent expression, thereby establishing a mechanism to achieve reproducibly higher levels of transgene transcription.
germ
to think of In “gain-offunction” mutations a functional gene is introduced into the germ line whose expression elicits a dominant phenotypic effect (Fig. 2A, Fig. 2B). In contrast, “loss-of-function” mu-
2996
although oversimplistic, as of two major types.
National
tAbbreviations:
ES cells, embryonic
tosidase gene; LCR, locus deficiency virus; HPRT,
control
region;
stem
cells; lacZ,
HIV,
human
13-galacimmuno-
hypoxanthine-guanine phosphoribosyitransferase; 1CM, embryonic inner cell mass; MDR1, multiple drug resistance gene; WBC, white blood cell.
0892-6638/91/0005-2996/$01.50.
© FASEB
ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe
Production
of Transgenic
hibits 13-galactosidase activity. The value of the trapped endogenous regulatory element or gene is demonstrated by the pattern of embryonic lacZ expression. Interesting gene sequences can then be cloned and characterized.
Micel
DONOR FEMALE
CREATION MUTATIONS
A
AND
CORRECTION
OF
GENETIC
Molecular genetics represents a powerful and versatile way to study gene function. Essential information has been obtained by correlating changes in gene activity with negative and positive gene mutations. Transgenic technology has greatly strengthened this approach in mammals by providing a system to create, identify, and characterize specific mutations.
B +virus
I.... I 8-CellEmbryos
N
‘I”
IGuided Tour I Monomer
of Genetic
I
Manipulationi I Multimerl
Augmentation
Trensgenlc house
+
different methods for generating transgenic mice are shown. Genetic modifications can be made in mouse embryos isolated from the reproductive tract of a donor female (top) at different developmental stages. Stages shown are 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, and blastocyst. A) Purified DNA fragments are microinjected into the pronucleus of 1-cell embryos. B) Preimplantation, cleavage-stage embryos are infected with retroviruses carrying foreign genetic information. C) Foreign DNA is inserted into ES cells, cloned from the inner cell mass (1CM) of the blastocyst, by electroporation or microinjection. ES cells possessing the new preferred genetic configuration are selected in vitro, and reintroduced into a recipient embryonic blastocoel, where they are Figure
1. Three
incorporated
into
the
1CM.
Embryos
reproductive tract of a foster mother velop to term.
are
transferred
(black arrows),
to
gain of function
dominsnt gof
(got)
Targeted
Correction
-c:::.J-
pj
-c:i--
E
_
(rare)
Interference
-cj--
MS
:J-j
lot N
dominant
Vnegative
+
the
where they de-
partial o’ got
I
+‘
‘I________
loss of function
Targeted
(rare)
Knockout
(lof)
It is also often observed that transgenic mice bearing intact genes are more likely to exhibit biologically appropriate patterns and levels of expression than transgenic mice bearing intronless cDNAs (12). Recently, a systematic approach has been used to determine the role of introns in transgene expression (13). This study shows that the presence of intervening sequences from a heterologous source can dramatically improve transgene expression. The reason for this is still not clear; however, the difference between genes and cDNAs may be attributed to the presence of controlling elements within the introns, an uncharacterized relationship between gene splicing and expression, or a contribution by intervening sequences to nucleosome phasing or some other higher order form of DNA structure. Further in vivo study of intron structure and function should lead to more precise, appropriate and predictable control of transgene expression. Finally, transgenic mice have been used to identify novel regulatory regions or genes active during development. Using enhancer-trapping, minimal promoters driving the bacterial f3-galactosidase reporter gene (lacZ) have been activated in cell- and tissue-specific patterns during the development of transgenic mice by chance integration near powerful endogenous control elements (see ref 14 for review). In genetrapping, the lacZ gene is placed downstream of a strong splice acceptor site, encouraging a splicing event to occur between the transgene and an upstream endogenous gene, resulting in the formation of a fusion gene product that ex-
TRANSGENIC
ANIMALS
IN RESEARCH
t #{176}
Functional
kYL
Inactive
___
Monomer
Multimer
Figure 2. A schematic dogenous gene expression
representation
of ways to manipulate
by the introduction
of foreign
DNA.
enAp-
proaches: A) augmentation (dominant positive); B) targeted correction; C) interference (dominant negative); D) targeted knockout. The endogenous gene is represented by a white rectangle, and a black circle within the gene indicates the presence of a mutation that inactivates gene function. Shown are genetic mutations that can “cure” (A, B) by establishing a gain-of-function (gof) or “cripple” (C, D) by causing a loss-of-function (lof). Foreign DNA can: integrate
randomly
as a fragment
containing
either
a cellular
or viral
promoter (black box) driving expression of the exogenous gene (A, C); or be designed to undergo homologous recombination as a gene fragment resembling the target endogenous gene (B, D). The phenotype of animals possessing these genotypic alterations will depend on whether the protein product of the modified gene normally functions
as an isolated
complex (i.e., tetramers for details.
monomer
(at left),
at right) (defined
or only
as a multimeric
in key at bottom).
See text
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The integration turb endogenous mutations
are
of foreign DNA into the genome gene structure and function. Loss-of
usually
recessive,
requiring
the
can perfunction
insertional
in-
activation of both alleles. The discovery and characterization of insertional mutations caused by the integration of transgenes and the subsequent isolation and sequencing of the disrupted endogenous gene have led to advancements in our understanding of development and genetic disease. Because insertion of retroviruses and microinjected DNA sequences is generally random, specific mutations are not chosen, but are instead generated by chance. Nevertheless, these techniques have been used to create dozens of recessive mutations, several of which map to previously characterized loci such as downless, limb deformity, and hotfoot (15-17). If a mutant phenotype could be selected in vitro, randomly generated mutations could be more efficiently used. Mutated ES cells have been selected for the presence or absence of a functional hypoxanthine-guanine phosphoribosyltransferase
(HPRT)
gene
before
being
used
to
generate
a
transgenic mouse model for Lesch-Nyhan syndrome (18, 19). Because the HPRT gene is located on the X chromosome, a single intragenic mutation can completely inactivate HPRT function in male-derived ES cells, resulting in a readily detectable phenotype. Unfortunately, most genes are neither X-linked nor selectable. Targeted knockout of nonselectable genes (Fig. 2D) was achieved only by the development of technology permitting the generation and detection of rare site-specific homologous recombination events in ES cells, including positive-negative selection and polymerase chain reaction (7). The parameters that govern the success of this technique have been thoroughly described elsewhere (7, 8). Genetically altered ES cells have been reintroduced into mouse embryos and allowed to colonize the germ line, causing heritable loss-of-function mutations. When homozygous transgenic mice are generated, the resulting phenotype demonstrates the function of the crippled endogenous gene (Fig. 2D). This approach has been used to achieve null mutations of the 132-microglobulin gene, resulting in the loss of major histocompatibility complex class 1 antigen and CD48 T cell-mediated cytotoxicity (20, 21); the Wnt-1 protooncogene, resulting in the abnormal development of midbrain and cerebellum (22, 23); the src protooncogene, resulting in osteopetrosis (24); and the Hox-1.5 gene, resulting in a constellation of phenotypic defects including abnormalities of the thyroid, parathyroid, thymus, salivary gland, heart, and the cartilage, bone, and musculature associated with the throat (25). Dominant mutations can also cause loss-of-function, if the presence of a mutated protein interferes with the function of the normal gene product. For example, by associating with normal endogenous proteins, an overexpressed aberrant subunit
can
inactivate
an
entire
multimeric
complex
(Fig.
2C). The most dramatic example of a dominant negative mutations is the induction of osteogenesis imperfecta in transgenic mice carrying a mutated a 1(I) collagen gene (26). Type I collagen functions as a triple-stranded helix, consisting of two a 1(I) chains and one a2(I) chain. The aberrant form of a 1(I) collagen binds to endogenous collagen subunits and alters the structure and function of the resulting helical complex, even when the mutated form is produced at 10% of the level of the endogenous protein (26). As another example, sickled erythrocytes form in transgenic mice overproducing human sickle hemoglobin (27, 28). This is a powerful and relatively simple genetic approach that will facilitate analysis of complex function by dominant perturbation. Another transgenic technique that has been successfully used to study function through perturbation is toxigenics 2998
Vol. 5
November
1991
(see ref 29 for review). Knowledge of developmental gene regulation is used to direct the expression of toxins or toxinconjugates to particular cell types at specific times. The ablation of selected cell populations is a powerful reverse genetic tool for determining the function of developing and differentiated cells, and may be useful for dissecting cell lineage. Transgenes can cure as well as cripple. Gene therapy, the correction of genetic disease by direct molecular treatment of a mutated gene, requires that the defective genetic information be replaced or supplemented with a normal functional gene. In gene augmentation (Fig. 2A), the expression of new genetic material that randomly integrates into the genome of a defective somatic or embryonic stem cell can complement the host mutation, thereby curing the disease. Unfortunately, the transgene will occasionally integrate into and inactivate a necessary host gene, or be negatively influenced by endogenous flanking sequences. Also, the expression of a functional gene product will not necessarily cure diseases caused by defective multimeric proteins; the presence of mutated subunits can efficiently inactivate most entire complexes (Fig. 2A). Nevertheless, this approach has been used to ameliorate the symptoms of some genetic diseases caused by underproduction of normal proteins. For example, shiverer (shi) mice transgenic for a normal mouse myelin basic protein gene produce compacted myelin in the central nervous system and no longer shiver (30); /3-thalasemia in mice homozygous for a deletion in the endogenous flrnai globin gene can be extinguished by introduction of a normal mouse 13maJ globin gene (31); and a gonadotropic-releasing hormone transgene can compensate for the hypogonadal (hpg) mutation in mice (32). A gain-of-function approach that is more powerful and free of potential genetic side-effects is targeted gene correction (Fig. 2B), in which defective DNA sequences are replaced with functional sequences without additional genetic disturbance. The best method for gene correction is by genetically manipulating cultured stem cells, such as ES cells or bone marrow cells, selecting for cells carrying a corrected allele and reintroducing those cells back into a suitable host organ (Fig. IC). Thompson et al. (33) corrected an HPRT-deficient ES cell line by targeted homologous recombination, and then produced a chimeric mouse in which the HPRT ES cells had contributed to the germ line. This approach has the potential to cure any genetic defect in animals; however, ethical issues may restrict genetic modification to various somatic stem cells in humans.
MODELS FOR THE DEVELOPMENT TREATMENT OF DISEASE
AND
One
of the most important applications of transgenic techis the creation of animal models of human disease. This potential has been realized by the extensive characterization of gene promoters and enhancers, permitting directed expression of foreign genes to specific types of cells. Once established, transgenic models can be used to dissect molecular mechanisms contributing to the pathogenesis of specific diseases, and to identify agents that can abrogate the onset of the disease, slow its progression, or ameliorate its symptoms. Transgenic mice have been used to establish models for AIDS and AIDS-related disorders. When the human immunodeficiency virus (HIV) transactivating tat gene is placed under the control of the HIV regulatory region, skin lesions appear resembling Kaposi’s sarcoma (34). A line of transgenic mice bearing the intact HIV provirus developed a spontaneous and fatal disease reminiscent of human AIDS (35). nology
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Recently the transgenic approach has provided new insight into cardiovascular homeostasis and disease. Overexpression of the mouse ren-2 transgene in the rat adrenal gland elicits severe hypertension (36). Ohkubo et al. (37) showed that production of rat renin together with angiotensinogen causes elevated systolic blood pressure in transgenic mice, which can be reversed by the angiotensinconverting enzyme inhibitor captopril. In contrast, transgenic mice expressing the atrial natriuretic factor in the liver exhibit hypotension (38). The role of oncogenes in tumorigenesis is being elucidated by targeting the expression of various types of oncogenes to specific tissues of transgenic animals. The dominant expression of activated oncogenes often results in the development of heritable tumors, clearly implicating these genes in oncogenesis. Several cogent and informative reviews have been written on this subject (1, 3, 5). I will focus on a few significant aspects of oncogenes in transgenic mice. It has been recognized for many years that specific chromosomal translocations are associated with distinct forms of cancer. Molecular analysis of the chromosomal breakpoints have established that protooncogenes are often relocated and mutated. These rearrangement events can be recreated in vivo by generating transgenic mice possessing similarly modified genes. Transgenic mice bearing a deregulated fusion minigene consisting of the bcl-2 protooncogene juxtaposed to the immunoglobulin heavy-chain gene [mimicking t(14;18)] exhibit follicular lymphoid hyperplasia that can progress to lymphoma (39). Mice transgenic for a bcr-abl fusion gene [mimicking the t(9;22) Philadelphia chromosome] demonstrate acute leukemia (40). These kinds of transgenic experiments validate the causal relationship between translocations and their associated cancers. Transgenic animals afford the opportunity to test the hypothesis that cancer progresses in a multistep fashion, requiring activation of a number of oncogenes. Transgenic mice bearing distinct oncogenes are derived independently and then cross-mated to generate doubly transgenic animals. The synergistic behavior of ras and myc has been demonstrated in mammary gland differentiation and tumorigenesis (41). Cooperativity has also been established between ras, myc, and SV4O T-antigen in hepatocarcinogenesis (42). Peptide growth factors have been implicated in both physiologic and pathologic processes for many years, and are now being examined in the context of the whole animal. Transforming growth factor-a, which triggers cellular proliferation by binding to and activating the epidermal growth factor receptor, has been overexpressed in a wide variety of transgenic mouse organs, resulting in a constellation of phenotypic alterations, including metaplasia of the pancreas, neoplasia of the mammary gland and liver, abnormal organogenesis of the eye, and hyperplasia and psoriasis of the skin (43-47). Transgenic mice overexpressing nerve growth factor in pancreatic islet cells exhibit a striking increase in islet innervation by a single subtype of sympathetic neuron (48). Transgenic animals overproducing cytokines and hemopoietic growth factors have also been described: interleukin-4 causes an inflammatory disease reminiscent of an allergic reaction and perturbs T cell development (49); interleukin-5 induces eosinophilia and stimulates the production of autoantibodies (50, 51); and granulocyte-macrophage colonystimulating factor stimulates the accumulation of macrophages, inducing lesions of the eye and striated muscle, and premature death (52). Transgenic mice hold great potential as test systems for the improvement of the treatment of disease. A compelling argument for the value of transgenic animals as preclinical in vivo
TRANSGENIC
ANIMALS
IN RESEARCH
models can be made in the field of drug resistance. A major obstacle to the treatment and eradication of human cancers is the ability of malignant cells to resist a broad spectrum of conventional chemotherapeutic agents. Cells can evade chemotherapy by overexpressing the cell-surface multidrug transporter P-glycoprotein, encoded by the multidrug resistance gene MDR1 (Fig. 3A). Mice transgenic for human MDR1 have been created that express P-glycoprotein in their bone marrow, which becomes resistant to the leukopenia induced in nontransgenic animals by chemotherapeutic drugs (53, 54). Significantly, bone marrow resistance in MDR1 mice is reversed by simultaneous administration of known multidrug transporter inhibitors such as quinidine (Fig. 3B, Fig. 3C). These mice should prove valuable as a rapid test system to determine the efficacy of anti-cancer agents, especially those that reverse multidrug resistance in animals. Another major site of chemotherapeu tic drug toxicity is the gastrointestinal tract. In a separate study, expression of a mutant dihydrofolate reductase transgene in the mouse intestine conveyed systemic resistance to methotrexate (55).
B IMultidrug
A
Resistant
Resistant
Transgenic Sensitive
wc
Micel WBC
.
-H-
t-
I
C
Daunomycin (10mg/kg)
80 U
*60
K
40 20
o
25
50
75 100 125 150
Ouinidine (mg/kg)
150 0 No Dau Non-MOR
Figure 3. Schematic (A, B) and graphic (C) depiction of multidrug resistance in transgenic mice. A) Multidrug-resistant, MDR1 transgenic mice possess white blood cells (WBC) expressing the multidrug transporter P-glycoprotein ( t) on the cell surface (dark line separates outside and inside of the cell). The anti-cancer drug daunomycin ( ‘ ) at the cell surface enters the WBC by pas-
sive diffusion,
but is expeditiously
pumped
out by the multidrug
transporter (white arrows). B) When the reversing agent quinidine (#{149}) is included, it competes for binding to the transporter and renders the cell daunomycin-sensitive. C) Dose-dependent reversal
of bone marrow agent quinidine.
protection against daunomycin by the reversing Daunomycin is administered to MDRI transgenic
mice by a single intraperitoneal injection, and WBC are counted S days later. Ordinate, percent of WBC count on day 5 compared with count before injection; ascissa, amount of quinidine coinjected; No Dau, quinidine alone; Non-MDR, daunomycin alone in nontransgenic control mice. C was taken in modified form from
ref 54.
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BIOTECHNOLOGY The advent of recombinant DNA technology has made it feasible to produce pharmacologically valuable proteins on an industrial scale. However, proteins synthesized in yeast and bacteria are not posttranslationally modified, often resulting in the reduction or elimination of activity. Mammalian tissue culture cells accurately modify these same proteins, but are expensive and technically difficult to maintain. Transgenic rabbits, sheep, and pigs provide viable alternatives to these other systems for the large-scale production of bioactive pharmaceutical proteins. Several excellent reviews are available on transgenics and biotechnology (2, 56, 57). The best-studied transgenic system for large-scale protein production is the mammary gland. The potential of the mammary gland as a bioreactor is being realized due to the cloning and characterization of genes encoding abundant milk proteins. Expression of genes of pharmaceutical interest can be targeted to the mammary gland by placing them under the control of the regulatory elements of genes encoding milk proteins such as the whey acidic protein, /3-lactoglobulin, and /3-casein. Pharmaceutically active proteins have been successfully expressed in mammalian milk, including human tissue plasminogen activator, ai-antitrypsin, coagulation factor IX, and interleukin 2 (see refs 56, 57 for review), and could be used to treat human diseases such as myocardial infarction, emphysema, and hemophilia. Unfortunately, although expression of these transgenes has been accurately directed, the level of production remains variable and generally low (57). The incorporation of yet to be identified milk protein gene-specific LCRs into the transgene might insulate transgenes from the influence of inhibiting flanking sequences and stimulate their activity. The agricultural industry is certain to benefit from improvements to livestock through genetic engineering. Domestic animals could be productively modified by introducing heritable mutations associated with growth, reproductive proficiency, or disease resistance. Overproduction of the bovine growth hormone gene in transgenic pigs was found to induce a significant daily weight gain, an enhancement in the efficiency of food conversion and a reduction in backfat (58). However, these transgenic pigs also exhibit a number of damaging lesions including arthritis, gastric ulcers, dermatitis, and renal disease, and die prematurely (58). Future refinements in temporal and spatial targeting by heterologous promoters should dramatically improve the current situation. CONCLUSIONS
AND
PROSPECTS
It is ironic indeed that after years of being forsaken by researchers preferring an in vitro or cell-free approach to the elucidation of scientific problems, the whole animal is once again in vogue as an experimental model system. The popularity of the transgenic approach is well deserved, for it is uniquely suited to the study of most complex biological systems. In this review I have described several exciting areas that are currently the beneficiaries of transgenic technology. The future will undoubtedly see continued progress as transgenesis is refined, resulting in significant advances at a number of fronts. The goal of achieving efficient, predictable, and insertion site-independent transgene expression will be actively pursued by continuing to elucidate the mechanisms regulating in vivo gene activity. Identification of LCRs and other master regulatory elements will benefit all transgenic fields, and the agricultural industry in particular. It is likely
that many technical and financial problems associated with genetic engineering of livestock and the industrial production of pharmaceuticals will be overcome by the identification of genetic elements that more precisely target transgene expression. The processes that govern the conversion of a single, fertilized egg to a multicellular organism are complex and obscure; techniques that can facilitate efficient gene targeting and the establishment of null mutations in the mammalian germ line are being used to construct a detailed developmental genetic map, which may ultimately revolutionize developmental biology. Transgenic animals will continue to serve as excellent models of a variety of human diseases. Finally, pioneering studies in transgenic animals have greatly enhanced the prospects for future gene therapy. Methodology is being refined using transgenic animals that will enable the selection of corrected genes in stem cells, which can “cure” when reintroduced into an appropriate organ of a host mammal, whether mouse or human. I would like to thank Drs. Paul Overbeek, Eric Chamelli Jhappan, Ira Pastan, and Michael Gottesman
Sandgren, for useful
discussions.
REFERENCES 1. Hanahan, D. (1988) Dissecting multistep tumorigenesis in transgenic mice. Annu. Rev. Genet. 22, 479-519 2. Jaenisch, R. (1988) Transgenic animals. Science 240, 1468-1474 3. Hanahan, D. (1989) Transgenic mice as probes into complex systems. Science 246, 1265-1275 4. Connelly, C. S., Fahl, W. E., and lannaccone, P. M. (1989) The role of transgenic animals in the analysis of various biological aspects of normal and pathologic states. Exp. Cell Res. 183, 257-276 5. Babinet, C., Morello, D., and Renard, J. P. (1989) Transgenic animals. Genome 31, 938-949 6. Hanahan, D. (1990) Transgenic mouse models of self-tolerance and autoreactivity by the immune system. Annu. Rev. Cell Bid. 6, 493-537 7. Capecchi, M. R. (1989) Altering the genome by homologous recombination. Science 244, 1288-1292 8. Frohman, M. A., and Martin, G. R. (1989) Cut, paste, and save: new approaches to altering specific genes in mice. Cell 56, 145-147 9. Hammer, R. E., Krumlauf, R., Camper, S. A., Brinster, R. L., and Tilghman, S. M. (1987) Diversity of alpha-fetoprotein gene expression in mice generated by a combination of separate enhancer elements. Science 235, 53-58 10. Townes, T. M., and Behringer, R. R. (1990) Human globin locus activation region (LAR): role in temporal control. Trends Genet. 6, 219-223 A., Winter,
11. Stief,
D. M.,
Stratling,
W. H.,
and
Sippel,
A. E.
(1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature (London) 341, 343-345 12. Brinster, R. L., Allen, J. M., Behringer, R. R., Gelinas, R. E., and Palmiter, R. D. (1988) Introns increase transcriptional efficiency in transgenic mice. Proc. NaiL Acad. Sci. USA 85, 836-840
13. Palmiter, R. D., Sandgren, E. D. D., and Brinster, R. L. (1991) hance expression of transgenes USA 88, 478-482 14. Rossant, J., and Joyner, A. L. genetic
analysis
of mammalian
P., Avarbock, M. R., Allen, Heterologous introns can enin mice. Proc. Nati. Aced. Sci. (1989) Towards development.
a molecularTrends Genet. 5,
27 7-283 15. Shawlot, W., Siciliano, M. J., Stallings, R. L., and Overbeek, P. A. (1989) Insertional inactivation of the downless gene in a family of transgenic mice. Mol. Biol. Med. 6, 299-307
3000 Vol. 5 November 1991 The FASEB Journal MERLINO ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe
16. Woychik, R. P., Maas, R. L., Zeller, R., Vogt, T. F., and Leder, P. (1990) ‘Formins’: proteins deduced from the alternative transcripts of the limb deformity gene. Nature (London) 346, 850-853 17. Gordon, J. W., Uehlinger, J., Dayani, N., Talansky, B. E., Gordon, M.,
Rudomen,
G. S., and
Neumann,
P. E. (1990)
Analysis
of the
hotfoot (ho) locus by creation of an insertional mutation in a transgenic mouse. Dev. Biol. 137, 349-358 18. Kuehn, M. R., Bradley, A., Robertson, E. J., and Evans, M. J. (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice. Nature (London) 326, 295-298 19. Hooper, M. L., Hardy, K., Handyside, A., Hunter, S., and Monk, M. (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature (London) 326, B. H., Marrack, P., Kappler, J. W., and Smithies, 0. (1990) Normal development of mice deficient in 2M, MHC class I proteins, and CD8 T cells. &ience 248, 1227-1230 21. Zijlstra, M., Bix, M., Simister, N. E., Loring,J. M., Raulet, D. H.,
R. (1990) 32-Microglobulin
deficient
24. 25. 26.
oncogene is required for development of a large region of the mouse brain. Cell 62, 1073-1085 Thomas, K. R., and Capecchi, M. R. (1990) Targeted disruption of the murine mt-i proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature (London) 346, 847-850 Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64, 693-702 Chisaka, 0., and Capecchi, M. R. (1991) Regionally restricted developmental defects resulting from targeted disruption of the mouse homebox gene hox-1.5. Nature (London) 350, 473-479 Stacey, A., Bateman, J., Choi, T, Mascara, T., Cole, W., and Jaenisch, R. (1988) Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineering gene. Nature (London) 332, 131-136
27.
mutant
pro-al(I)
collagen
Ryan, T. M., Townes, T M., Reilly, M. P., Asakura, T., Palmiter, R. D., Brinster, R. L., and Behringer, R. R. (1990) Human sickle hemoglobin
in transgenic
mice.
Science 247,
566-568
28. Greaves, D. R., Fraser, P., Vidal, M. A., Hedges, M. J., Ropers, D., Luzzatto, L., and Grosveld, F. (1990) A transgenic mouse model of sickle cell disorder. Nature (London) 343, 183-185 29. Evans, G. A. (1989) Dissecting mouse development with toxigenics. Genes Dcv. 3, 259-263 30. Readhead, C., Popko, B., Takahashi, N., Shine, H. D., Saavedra, R. A., Sidman, R. L., and Hood, L. (1987) Expression of a myelin basic
protein
gene
in transgenic
shiverer
mice:
correction
of the
dysmyelinating phenotype. Cell 48, 703-712 31. Costantini, F., Chada, K., and Magram, J. (1986) Correction of murine $-thalassemia by gene transfer into the germ line. &ience 233, 1192-1194 32. Mason, A. J., Pitts, S. L., Nikolics, K., Szonyi, E., Wilcox, J. N., Seeburg, P. H., and Stewart, T A. (1986) The hypogonadal mouse: reproductive functions restored by gene therapy. Science 234, 1372-1378
lymphoid transgenic
hyperplasia to high-grade malignant lymphoma in mice for the t(14;18). Nature (London) 349, 254-256 40. Heisterkamp, N., Jenster, G., ten Hoeve, J., Zovich, D., Pattengale, P. K., and Groffen, J. (1990) Acute leukemia in bcr/abl transgenic mice. Nature (London) 344, 251-253 41. Pattengale, P. K., Stewart, T. A., Leder, A., Sinn, E., Muller, W., Tepler, I., Schmidt, E., and Leder, P. (1989) Animal models of human disease: pathology and molecular biology of spontaneous ne-
oplasms occurring vated
in transgenic mice carrying and expressing actiAm. j Pathol. 135, 39-61 E. P., Quaife, C. J., Pinkert, C. A., Palm iter, R. D., and
cellular
Brinster,
oncogenes.
R. L. (1989)
Oncogene-induced
liver neoplasia
in trans-
genic mice. Oncogene 4, 715-724 43. Jhappan, C., Stahle, C., Harkins, R. N., Fausto, N., Smith, G. H., and Merlino, G. T. (1990) TGFx overexpression in transgenic mice
mice lack
CD48 cytolytic T cells. Nature (London) 344, 742-746 22. McMahon, A. P., and Bradley, A. (1990) The Wnt-1 (mt-i) proto-
23.
M. E., Cochrane, K. L., and Field, L. J. (1990) in transgenic mice expressing atrial natriuretic factor fusion genes. Hypertencion 16, 301-307 39. McDonnell, T. J., and Korsmeyer, S. J. (1991) Progression from
42. Sandgren,
292-295
20. Koller,
and Jaenisch,
38. Steinhelper, Hypotension
induces liver neoplasia and abnormal development mary gland and pancreas. Cell 61, 1137-1146
of the mam-
44. Sandgren, E. P., Luetteke, N. C., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1990) Overexpression of 1tFa in transgenic mice: induction
of epithelial
hyperplasia,
pancreatic
metaplasia,
and car-
cinoma of the breast. Cell 61, 1121-1135 45. Matsui, Y., Halter, S. A., Holt,J. T., Hogan, B. L. M., and Coffey, R. J. (1990) Development of mammary hyperplasia and neoplasia in MMTV-TGFa transgenic mice. Cell 61, 1147-1155 46. Vassar, R., and Fuchs, E. (1991) Transgenic mice provide new insights into the role of TGFcs during epidermal development and differentiation. Genes Dcv. .5, 714-727 47.
Silversides, D. W., Oosterloo, M. -L., Merin, L. M., Derynck, R., Ullrich, A., and Overbeek, P. A. (1991) TGFs and EGF transgenes alter developmental morphogenesis of the mouse eye. Development In
press 48. Edwards, R. H., Rutter, W. J., and Hanahan, D. (1989) Directed expression of NGF to pancreatic cells in transgenic mice leads to selective hyperinnervation of the islets. Cell 58, 161-170 49. Tepper, R. I., Levinson, D. A., Stanger, B. Z., Campos-Torres, J., Abbas, A. K., and Leder, P. (1990) IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice. Cell 62, 457-467 50. Dent, L. A., Strath, M., Mellor, A. L., and Sanderson, C. J. (1990) Eisoinophilia in transgenic mice expressing interleukin 5. j Exp. Med. 172, 1425-1431 51. Tominaga, A., Takaki, S., Koyama, N., Katoh, S., Matsumoto, R., Migita, M., Hitoshi, Y., Hosoya, Y., Yamauchi, S., Kanai, Y., Miyazaki, J. -I., Usuku, G., Yamamura, K. -I., and Takatsu, K. (1991) Transgenic mice expressing a B cell growth and differentiation factor gene (interleukin 5) develop eosinophilia and autoantibody production. j Exp. Med. 173, 429-437 52. Lang, R. A., Metcalf, D., Cuthbertson, R. A., Lyons, I., Stanley, E., Kelso, A., Kannourakis, G., Williamson, D. J., Klintworth, G. K., Gonda, T J., and Dunn, A. R. (1987) Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage. Cell. 51, 675-686 53. Galski, H., Sullivan, M., Willingham, M. C., Chin, K. -V., Gottesman, M. M., Pastan, I., and Merlino, G. T. (1989) Expression of a human multidrug resistance eDNA (MDR 1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia. Mol. CelL Biol. 9, 4357-4363 54. Mickisch, G. H., Merlino, G. T., Galski, H., Gottesman, M. M., and Pastan, I. (1991) Transgenic mice that express the multidrugresistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance. Proc. NaiL Aced. Sci. USA 88,
33. Thompson, S., Clarke, A. R., Pow, A. M., Hooper, M. L., and Melton, D. W. (1989) Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56, 313-321 34. Vogel, J., Hinrichs, S. H., Reynolds, R. K., Luciw, P. A., and Jay, G. (1988) The HIV tat gene induces dermal lesions resembling Kaposi’s sarcoma in traisgenic mice. Nature (London) 335, 606-611 35. Leonard, J. M., Abramczuk, J. W., Pezen, D. S., Rutledge, R., Belcher, J. H., Hakim, F., Shearer, G., Lamperth, L., Travis, W., Fredrickson, T., Notkins, A. L., and Martin, M. A. (1988) Develop547-551 ment of disease and virus recovery in transgenic mice containing 55. Isola, L. M., and Gordon, J. W. (1989) Expression of a methotrexHIV proviral DNA. Science 242, 1665-1670 ate resistant dihydrofolate reductase gene in transgenic mice. Dcv. 36. Mullins, J. J., Peters, J., and Ganten, D. (1990) Fulminant hyperGen. 10, 349-355 tension in transgenic rats harbouring the mouse Ren-2 gene. Nature 56. Westphal, H. (1989) Transgenic mammals and biotechnology. (London) 344, 541-544 FASEBJ 3, 117-120 37. Ohkubo, H., Kawakami, H., Kakehi, Y., Takumi, T, Arai, H., L. (1990) The mammary gland as a bioreactor: Yokota, Y., Iwai, M., Tanabe, Y., Masu, M., Hata, J., Iwao, H., 57. Hennighausen, production of foreign proteins in milk. Prot. Exp. Purzf 1, 3-8 Okamoto, H., Yokoyama, M., Nomura, T., Katsuki, M., and Nakanishi, S. (1990) Generation of transgenic mice with elevated 58. Pursel, V. G., Pinkert, C. A., Miller, K. F., Bolt, D. J., Campbell, blood pressure by introduction of the rat renin and angiotensinogen R. G., Palmiter, R. D., Brinster, R. L., and Hammer, R. E. (1989) genes. Proc. NatL Acad. Sci. USA 87, 5153-5157 Genetic engineering of livestock. Science 244, 1281-1288
3001 TRANSGENIC ANIMALS IN RESEARCH ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe
animals
GLENN
in biomedical
research
T. MERLINO
Laboratory Maryland
of Molecular 20892, USA
Biology,
National
Cancer
Institute,
The advent of transgenic technology, in which foreign genetic information is stably introduced into the mammalian germ line, has dramatically enhanced our basic knowledge of physiologic and pathologic processes. Transgenic animals created by these genetic manipulations are being used to provide insights into gene regulation, development, pathogenesis, and the treatment of disease. Furthermore, transgenic biotechnology holds great promise for the creation of genetically superior livestock and the industrial production of precious pharmaceuticals. It is evident now that the study and use of transgenic animals will significantly improve the human condition. Merlino, G. T. Transgenic animals in biomedical research. FASEB J. 5: 2996-3001; 1991. ABSTRACT
Key Words: biotechnology
.
gene regulation . gene therapy targeted gene inactivation
.
cancer
.
mutation
ABILITY TO PRECISELY AND PERMANENTLY modify genetic information by introducing foreign DNA into the germ line of animals has elevated the potential of biological research to an unprecedented level. This review describes how the study of transgenic animals has contributed to our ability to understand gene regulation, development, and the molecular basis of disease, and has facilitated improvements in gene therapy and biotechnology. This field has been explosive in its growth, and hundreds of reports are published yearly describing the generation, characterization, and use of transgenic animals. It is beyond the scope of this review to recount all this work, and therefore I have not discussed or cited many important contributions, as well as entire fields such as immunology; however, several excellent and comprehensive reviews of specialized areas are available and can be consulted (1-6). Instead, I will attempt to instill an understanding and appreciation of basic principles, describe recent developments, and speculate on future trends. Three main methods have been used to transfer genes into
THE
the
mouse
germ
line.
The
most
efficient
and
commonly
used
technique is the direct microinjection of DNA fragments into the pronuclei of one-cell embryos, which are then returned to the oviduct of a foster mother to complete development (Fig. 1A). In a less arduous method, cleavage-stage embryos are exposed to recombinant retroviruses in vitro and are returned to the uterus of a foster mother (Fig. 1B). Finally, recent technical advancements have permitted the in vitro maintenance, genetic manipulation, and selection of pleuripotent embryonic stem (ES)1 cells derived from the inner cell mass (Fig. 1C). Upon reintroduction into the embryonic blastocoel, ES cells that have been subjected to gene manipulation
can
colonize
the
line (7, 8). It is useful, genetic manipulations
embryo
and
contribute
to the
Institutes
of Health,
Bethesda,
tations
are often inactivation of an eign DNA (Fig. method available
recessive and are usually the results of the endogenous gene by the integration of for2D). Gene targeting is the most efficient to produce loss-of-function mutations.
REGULATION
OF
GENE
EXPRESSION
Gene regulation has been examined extensively in vitro by expressing foreign gene constructs in cultured cells. However, the relevance of many cell culture studies to actual in vivo events is tenuous. Transgenic animals provide a true in vivo environment for evaluating the mechanisms by which gene expression is modulated in the adult and during development. Detailed analysis of myriad gene regulatory elements (promoters and enhancers) in the context of the whole animal has led to remarkable discoveries about gene function, and also to the development of more sophisticated tools for targeting the expression of heterologous genes to specific types of cells (2, 4). For example, regulatory elements that control the complex tissue- and stage-specific pattern of a-fetoprotein expression during mouse embryogenesis have been identified by reproducing their activity in transgenic mice (9). Transgenic mice have also been used to identify regulatory control elements that are localized a great distance from the coding region of the gene. In the human 13-globin locus distant control elements, called locus control regions (LCR) or locus activation regions, are responsible for maximum appropriate expression in transgenic mice (see ref 10 for review). In the absence of specific DNase I hypersensitive sites in the LCR region, /3-globin expression is reduced approximately 300-fold in transgenic mice. These regions may serve as nuclear matrix attachment sites, as binding sites for topoisomerase II or as erythroid-specific enhancers of replication (10). A serious problem in achieving efficient and predictable transgene expression is the potentially deleterious effect of endogenous genomic sequences near the site of integration. The presence of LCRs within transgenes may overcome the influence of these flanking sequences. Specific regions of mammalian DNA, A-elements, have been identified which mediate attachment of the chromatin to the nuclear scaffold (11). Reporter genes flanked by A-elements exhibit elevated expression that is insertion site-independent in stably transfected cultured cells. The availability of control elements that would render a transgene free from the unpredictable influences of the mouse genome would enable copy numberdependent expression, thereby establishing a mechanism to achieve reproducibly higher levels of transgene transcription.
germ
to think of In “gain-offunction” mutations a functional gene is introduced into the germ line whose expression elicits a dominant phenotypic effect (Fig. 2A, Fig. 2B). In contrast, “loss-of-function” mu-
2996
although oversimplistic, as of two major types.
National
tAbbreviations:
ES cells, embryonic
tosidase gene; LCR, locus deficiency virus; HPRT,
control
region;
stem
cells; lacZ,
HIV,
human
13-galacimmuno-
hypoxanthine-guanine phosphoribosyitransferase; 1CM, embryonic inner cell mass; MDR1, multiple drug resistance gene; WBC, white blood cell.
0892-6638/91/0005-2996/$01.50.
© FASEB
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Production
of Transgenic
hibits 13-galactosidase activity. The value of the trapped endogenous regulatory element or gene is demonstrated by the pattern of embryonic lacZ expression. Interesting gene sequences can then be cloned and characterized.
Micel
DONOR FEMALE
CREATION MUTATIONS
A
AND
CORRECTION
OF
GENETIC
Molecular genetics represents a powerful and versatile way to study gene function. Essential information has been obtained by correlating changes in gene activity with negative and positive gene mutations. Transgenic technology has greatly strengthened this approach in mammals by providing a system to create, identify, and characterize specific mutations.
B +virus
I.... I 8-CellEmbryos
N
‘I”
IGuided Tour I Monomer
of Genetic
I
Manipulationi I Multimerl
Augmentation
Trensgenlc house
+
different methods for generating transgenic mice are shown. Genetic modifications can be made in mouse embryos isolated from the reproductive tract of a donor female (top) at different developmental stages. Stages shown are 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, and blastocyst. A) Purified DNA fragments are microinjected into the pronucleus of 1-cell embryos. B) Preimplantation, cleavage-stage embryos are infected with retroviruses carrying foreign genetic information. C) Foreign DNA is inserted into ES cells, cloned from the inner cell mass (1CM) of the blastocyst, by electroporation or microinjection. ES cells possessing the new preferred genetic configuration are selected in vitro, and reintroduced into a recipient embryonic blastocoel, where they are Figure
1. Three
incorporated
into
the
1CM.
Embryos
reproductive tract of a foster mother velop to term.
are
transferred
(black arrows),
to
gain of function
dominsnt gof
(got)
Targeted
Correction
-c:::.J-
pj
-c:i--
E
_
(rare)
Interference
-cj--
MS
:J-j
lot N
dominant
Vnegative
+
the
where they de-
partial o’ got
I
+‘
‘I________
loss of function
Targeted
(rare)
Knockout
(lof)
It is also often observed that transgenic mice bearing intact genes are more likely to exhibit biologically appropriate patterns and levels of expression than transgenic mice bearing intronless cDNAs (12). Recently, a systematic approach has been used to determine the role of introns in transgene expression (13). This study shows that the presence of intervening sequences from a heterologous source can dramatically improve transgene expression. The reason for this is still not clear; however, the difference between genes and cDNAs may be attributed to the presence of controlling elements within the introns, an uncharacterized relationship between gene splicing and expression, or a contribution by intervening sequences to nucleosome phasing or some other higher order form of DNA structure. Further in vivo study of intron structure and function should lead to more precise, appropriate and predictable control of transgene expression. Finally, transgenic mice have been used to identify novel regulatory regions or genes active during development. Using enhancer-trapping, minimal promoters driving the bacterial f3-galactosidase reporter gene (lacZ) have been activated in cell- and tissue-specific patterns during the development of transgenic mice by chance integration near powerful endogenous control elements (see ref 14 for review). In genetrapping, the lacZ gene is placed downstream of a strong splice acceptor site, encouraging a splicing event to occur between the transgene and an upstream endogenous gene, resulting in the formation of a fusion gene product that ex-
TRANSGENIC
ANIMALS
IN RESEARCH
t #{176}
Functional
kYL
Inactive
___
Monomer
Multimer
Figure 2. A schematic dogenous gene expression
representation
of ways to manipulate
by the introduction
of foreign
DNA.
enAp-
proaches: A) augmentation (dominant positive); B) targeted correction; C) interference (dominant negative); D) targeted knockout. The endogenous gene is represented by a white rectangle, and a black circle within the gene indicates the presence of a mutation that inactivates gene function. Shown are genetic mutations that can “cure” (A, B) by establishing a gain-of-function (gof) or “cripple” (C, D) by causing a loss-of-function (lof). Foreign DNA can: integrate
randomly
as a fragment
containing
either
a cellular
or viral
promoter (black box) driving expression of the exogenous gene (A, C); or be designed to undergo homologous recombination as a gene fragment resembling the target endogenous gene (B, D). The phenotype of animals possessing these genotypic alterations will depend on whether the protein product of the modified gene normally functions
as an isolated
complex (i.e., tetramers for details.
monomer
(at left),
at right) (defined
or only
as a multimeric
in key at bottom).
See text
2997
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The integration turb endogenous mutations
are
of foreign DNA into the genome gene structure and function. Loss-of
usually
recessive,
requiring
the
can perfunction
insertional
in-
activation of both alleles. The discovery and characterization of insertional mutations caused by the integration of transgenes and the subsequent isolation and sequencing of the disrupted endogenous gene have led to advancements in our understanding of development and genetic disease. Because insertion of retroviruses and microinjected DNA sequences is generally random, specific mutations are not chosen, but are instead generated by chance. Nevertheless, these techniques have been used to create dozens of recessive mutations, several of which map to previously characterized loci such as downless, limb deformity, and hotfoot (15-17). If a mutant phenotype could be selected in vitro, randomly generated mutations could be more efficiently used. Mutated ES cells have been selected for the presence or absence of a functional hypoxanthine-guanine phosphoribosyltransferase
(HPRT)
gene
before
being
used
to
generate
a
transgenic mouse model for Lesch-Nyhan syndrome (18, 19). Because the HPRT gene is located on the X chromosome, a single intragenic mutation can completely inactivate HPRT function in male-derived ES cells, resulting in a readily detectable phenotype. Unfortunately, most genes are neither X-linked nor selectable. Targeted knockout of nonselectable genes (Fig. 2D) was achieved only by the development of technology permitting the generation and detection of rare site-specific homologous recombination events in ES cells, including positive-negative selection and polymerase chain reaction (7). The parameters that govern the success of this technique have been thoroughly described elsewhere (7, 8). Genetically altered ES cells have been reintroduced into mouse embryos and allowed to colonize the germ line, causing heritable loss-of-function mutations. When homozygous transgenic mice are generated, the resulting phenotype demonstrates the function of the crippled endogenous gene (Fig. 2D). This approach has been used to achieve null mutations of the 132-microglobulin gene, resulting in the loss of major histocompatibility complex class 1 antigen and CD48 T cell-mediated cytotoxicity (20, 21); the Wnt-1 protooncogene, resulting in the abnormal development of midbrain and cerebellum (22, 23); the src protooncogene, resulting in osteopetrosis (24); and the Hox-1.5 gene, resulting in a constellation of phenotypic defects including abnormalities of the thyroid, parathyroid, thymus, salivary gland, heart, and the cartilage, bone, and musculature associated with the throat (25). Dominant mutations can also cause loss-of-function, if the presence of a mutated protein interferes with the function of the normal gene product. For example, by associating with normal endogenous proteins, an overexpressed aberrant subunit
can
inactivate
an
entire
multimeric
complex
(Fig.
2C). The most dramatic example of a dominant negative mutations is the induction of osteogenesis imperfecta in transgenic mice carrying a mutated a 1(I) collagen gene (26). Type I collagen functions as a triple-stranded helix, consisting of two a 1(I) chains and one a2(I) chain. The aberrant form of a 1(I) collagen binds to endogenous collagen subunits and alters the structure and function of the resulting helical complex, even when the mutated form is produced at 10% of the level of the endogenous protein (26). As another example, sickled erythrocytes form in transgenic mice overproducing human sickle hemoglobin (27, 28). This is a powerful and relatively simple genetic approach that will facilitate analysis of complex function by dominant perturbation. Another transgenic technique that has been successfully used to study function through perturbation is toxigenics 2998
Vol. 5
November
1991
(see ref 29 for review). Knowledge of developmental gene regulation is used to direct the expression of toxins or toxinconjugates to particular cell types at specific times. The ablation of selected cell populations is a powerful reverse genetic tool for determining the function of developing and differentiated cells, and may be useful for dissecting cell lineage. Transgenes can cure as well as cripple. Gene therapy, the correction of genetic disease by direct molecular treatment of a mutated gene, requires that the defective genetic information be replaced or supplemented with a normal functional gene. In gene augmentation (Fig. 2A), the expression of new genetic material that randomly integrates into the genome of a defective somatic or embryonic stem cell can complement the host mutation, thereby curing the disease. Unfortunately, the transgene will occasionally integrate into and inactivate a necessary host gene, or be negatively influenced by endogenous flanking sequences. Also, the expression of a functional gene product will not necessarily cure diseases caused by defective multimeric proteins; the presence of mutated subunits can efficiently inactivate most entire complexes (Fig. 2A). Nevertheless, this approach has been used to ameliorate the symptoms of some genetic diseases caused by underproduction of normal proteins. For example, shiverer (shi) mice transgenic for a normal mouse myelin basic protein gene produce compacted myelin in the central nervous system and no longer shiver (30); /3-thalasemia in mice homozygous for a deletion in the endogenous flrnai globin gene can be extinguished by introduction of a normal mouse 13maJ globin gene (31); and a gonadotropic-releasing hormone transgene can compensate for the hypogonadal (hpg) mutation in mice (32). A gain-of-function approach that is more powerful and free of potential genetic side-effects is targeted gene correction (Fig. 2B), in which defective DNA sequences are replaced with functional sequences without additional genetic disturbance. The best method for gene correction is by genetically manipulating cultured stem cells, such as ES cells or bone marrow cells, selecting for cells carrying a corrected allele and reintroducing those cells back into a suitable host organ (Fig. IC). Thompson et al. (33) corrected an HPRT-deficient ES cell line by targeted homologous recombination, and then produced a chimeric mouse in which the HPRT ES cells had contributed to the germ line. This approach has the potential to cure any genetic defect in animals; however, ethical issues may restrict genetic modification to various somatic stem cells in humans.
MODELS FOR THE DEVELOPMENT TREATMENT OF DISEASE
AND
One
of the most important applications of transgenic techis the creation of animal models of human disease. This potential has been realized by the extensive characterization of gene promoters and enhancers, permitting directed expression of foreign genes to specific types of cells. Once established, transgenic models can be used to dissect molecular mechanisms contributing to the pathogenesis of specific diseases, and to identify agents that can abrogate the onset of the disease, slow its progression, or ameliorate its symptoms. Transgenic mice have been used to establish models for AIDS and AIDS-related disorders. When the human immunodeficiency virus (HIV) transactivating tat gene is placed under the control of the HIV regulatory region, skin lesions appear resembling Kaposi’s sarcoma (34). A line of transgenic mice bearing the intact HIV provirus developed a spontaneous and fatal disease reminiscent of human AIDS (35). nology
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MERLINO
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Recently the transgenic approach has provided new insight into cardiovascular homeostasis and disease. Overexpression of the mouse ren-2 transgene in the rat adrenal gland elicits severe hypertension (36). Ohkubo et al. (37) showed that production of rat renin together with angiotensinogen causes elevated systolic blood pressure in transgenic mice, which can be reversed by the angiotensinconverting enzyme inhibitor captopril. In contrast, transgenic mice expressing the atrial natriuretic factor in the liver exhibit hypotension (38). The role of oncogenes in tumorigenesis is being elucidated by targeting the expression of various types of oncogenes to specific tissues of transgenic animals. The dominant expression of activated oncogenes often results in the development of heritable tumors, clearly implicating these genes in oncogenesis. Several cogent and informative reviews have been written on this subject (1, 3, 5). I will focus on a few significant aspects of oncogenes in transgenic mice. It has been recognized for many years that specific chromosomal translocations are associated with distinct forms of cancer. Molecular analysis of the chromosomal breakpoints have established that protooncogenes are often relocated and mutated. These rearrangement events can be recreated in vivo by generating transgenic mice possessing similarly modified genes. Transgenic mice bearing a deregulated fusion minigene consisting of the bcl-2 protooncogene juxtaposed to the immunoglobulin heavy-chain gene [mimicking t(14;18)] exhibit follicular lymphoid hyperplasia that can progress to lymphoma (39). Mice transgenic for a bcr-abl fusion gene [mimicking the t(9;22) Philadelphia chromosome] demonstrate acute leukemia (40). These kinds of transgenic experiments validate the causal relationship between translocations and their associated cancers. Transgenic animals afford the opportunity to test the hypothesis that cancer progresses in a multistep fashion, requiring activation of a number of oncogenes. Transgenic mice bearing distinct oncogenes are derived independently and then cross-mated to generate doubly transgenic animals. The synergistic behavior of ras and myc has been demonstrated in mammary gland differentiation and tumorigenesis (41). Cooperativity has also been established between ras, myc, and SV4O T-antigen in hepatocarcinogenesis (42). Peptide growth factors have been implicated in both physiologic and pathologic processes for many years, and are now being examined in the context of the whole animal. Transforming growth factor-a, which triggers cellular proliferation by binding to and activating the epidermal growth factor receptor, has been overexpressed in a wide variety of transgenic mouse organs, resulting in a constellation of phenotypic alterations, including metaplasia of the pancreas, neoplasia of the mammary gland and liver, abnormal organogenesis of the eye, and hyperplasia and psoriasis of the skin (43-47). Transgenic mice overexpressing nerve growth factor in pancreatic islet cells exhibit a striking increase in islet innervation by a single subtype of sympathetic neuron (48). Transgenic animals overproducing cytokines and hemopoietic growth factors have also been described: interleukin-4 causes an inflammatory disease reminiscent of an allergic reaction and perturbs T cell development (49); interleukin-5 induces eosinophilia and stimulates the production of autoantibodies (50, 51); and granulocyte-macrophage colonystimulating factor stimulates the accumulation of macrophages, inducing lesions of the eye and striated muscle, and premature death (52). Transgenic mice hold great potential as test systems for the improvement of the treatment of disease. A compelling argument for the value of transgenic animals as preclinical in vivo
TRANSGENIC
ANIMALS
IN RESEARCH
models can be made in the field of drug resistance. A major obstacle to the treatment and eradication of human cancers is the ability of malignant cells to resist a broad spectrum of conventional chemotherapeutic agents. Cells can evade chemotherapy by overexpressing the cell-surface multidrug transporter P-glycoprotein, encoded by the multidrug resistance gene MDR1 (Fig. 3A). Mice transgenic for human MDR1 have been created that express P-glycoprotein in their bone marrow, which becomes resistant to the leukopenia induced in nontransgenic animals by chemotherapeutic drugs (53, 54). Significantly, bone marrow resistance in MDR1 mice is reversed by simultaneous administration of known multidrug transporter inhibitors such as quinidine (Fig. 3B, Fig. 3C). These mice should prove valuable as a rapid test system to determine the efficacy of anti-cancer agents, especially those that reverse multidrug resistance in animals. Another major site of chemotherapeu tic drug toxicity is the gastrointestinal tract. In a separate study, expression of a mutant dihydrofolate reductase transgene in the mouse intestine conveyed systemic resistance to methotrexate (55).
B IMultidrug
A
Resistant
Resistant
Transgenic Sensitive
wc
Micel WBC
.
-H-
t-
I
C
Daunomycin (10mg/kg)
80 U
*60
K
40 20
o
25
50
75 100 125 150
Ouinidine (mg/kg)
150 0 No Dau Non-MOR
Figure 3. Schematic (A, B) and graphic (C) depiction of multidrug resistance in transgenic mice. A) Multidrug-resistant, MDR1 transgenic mice possess white blood cells (WBC) expressing the multidrug transporter P-glycoprotein ( t) on the cell surface (dark line separates outside and inside of the cell). The anti-cancer drug daunomycin ( ‘ ) at the cell surface enters the WBC by pas-
sive diffusion,
but is expeditiously
pumped
out by the multidrug
transporter (white arrows). B) When the reversing agent quinidine (#{149}) is included, it competes for binding to the transporter and renders the cell daunomycin-sensitive. C) Dose-dependent reversal
of bone marrow agent quinidine.
protection against daunomycin by the reversing Daunomycin is administered to MDRI transgenic
mice by a single intraperitoneal injection, and WBC are counted S days later. Ordinate, percent of WBC count on day 5 compared with count before injection; ascissa, amount of quinidine coinjected; No Dau, quinidine alone; Non-MDR, daunomycin alone in nontransgenic control mice. C was taken in modified form from
ref 54.
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BIOTECHNOLOGY The advent of recombinant DNA technology has made it feasible to produce pharmacologically valuable proteins on an industrial scale. However, proteins synthesized in yeast and bacteria are not posttranslationally modified, often resulting in the reduction or elimination of activity. Mammalian tissue culture cells accurately modify these same proteins, but are expensive and technically difficult to maintain. Transgenic rabbits, sheep, and pigs provide viable alternatives to these other systems for the large-scale production of bioactive pharmaceutical proteins. Several excellent reviews are available on transgenics and biotechnology (2, 56, 57). The best-studied transgenic system for large-scale protein production is the mammary gland. The potential of the mammary gland as a bioreactor is being realized due to the cloning and characterization of genes encoding abundant milk proteins. Expression of genes of pharmaceutical interest can be targeted to the mammary gland by placing them under the control of the regulatory elements of genes encoding milk proteins such as the whey acidic protein, /3-lactoglobulin, and /3-casein. Pharmaceutically active proteins have been successfully expressed in mammalian milk, including human tissue plasminogen activator, ai-antitrypsin, coagulation factor IX, and interleukin 2 (see refs 56, 57 for review), and could be used to treat human diseases such as myocardial infarction, emphysema, and hemophilia. Unfortunately, although expression of these transgenes has been accurately directed, the level of production remains variable and generally low (57). The incorporation of yet to be identified milk protein gene-specific LCRs into the transgene might insulate transgenes from the influence of inhibiting flanking sequences and stimulate their activity. The agricultural industry is certain to benefit from improvements to livestock through genetic engineering. Domestic animals could be productively modified by introducing heritable mutations associated with growth, reproductive proficiency, or disease resistance. Overproduction of the bovine growth hormone gene in transgenic pigs was found to induce a significant daily weight gain, an enhancement in the efficiency of food conversion and a reduction in backfat (58). However, these transgenic pigs also exhibit a number of damaging lesions including arthritis, gastric ulcers, dermatitis, and renal disease, and die prematurely (58). Future refinements in temporal and spatial targeting by heterologous promoters should dramatically improve the current situation. CONCLUSIONS
AND
PROSPECTS
It is ironic indeed that after years of being forsaken by researchers preferring an in vitro or cell-free approach to the elucidation of scientific problems, the whole animal is once again in vogue as an experimental model system. The popularity of the transgenic approach is well deserved, for it is uniquely suited to the study of most complex biological systems. In this review I have described several exciting areas that are currently the beneficiaries of transgenic technology. The future will undoubtedly see continued progress as transgenesis is refined, resulting in significant advances at a number of fronts. The goal of achieving efficient, predictable, and insertion site-independent transgene expression will be actively pursued by continuing to elucidate the mechanisms regulating in vivo gene activity. Identification of LCRs and other master regulatory elements will benefit all transgenic fields, and the agricultural industry in particular. It is likely
that many technical and financial problems associated with genetic engineering of livestock and the industrial production of pharmaceuticals will be overcome by the identification of genetic elements that more precisely target transgene expression. The processes that govern the conversion of a single, fertilized egg to a multicellular organism are complex and obscure; techniques that can facilitate efficient gene targeting and the establishment of null mutations in the mammalian germ line are being used to construct a detailed developmental genetic map, which may ultimately revolutionize developmental biology. Transgenic animals will continue to serve as excellent models of a variety of human diseases. Finally, pioneering studies in transgenic animals have greatly enhanced the prospects for future gene therapy. Methodology is being refined using transgenic animals that will enable the selection of corrected genes in stem cells, which can “cure” when reintroduced into an appropriate organ of a host mammal, whether mouse or human. I would like to thank Drs. Paul Overbeek, Eric Chamelli Jhappan, Ira Pastan, and Michael Gottesman
Sandgren, for useful
discussions.
REFERENCES 1. Hanahan, D. (1988) Dissecting multistep tumorigenesis in transgenic mice. Annu. Rev. Genet. 22, 479-519 2. Jaenisch, R. (1988) Transgenic animals. Science 240, 1468-1474 3. Hanahan, D. (1989) Transgenic mice as probes into complex systems. Science 246, 1265-1275 4. Connelly, C. S., Fahl, W. E., and lannaccone, P. M. (1989) The role of transgenic animals in the analysis of various biological aspects of normal and pathologic states. Exp. Cell Res. 183, 257-276 5. Babinet, C., Morello, D., and Renard, J. P. (1989) Transgenic animals. Genome 31, 938-949 6. Hanahan, D. (1990) Transgenic mouse models of self-tolerance and autoreactivity by the immune system. Annu. Rev. Cell Bid. 6, 493-537 7. Capecchi, M. R. (1989) Altering the genome by homologous recombination. Science 244, 1288-1292 8. Frohman, M. A., and Martin, G. R. (1989) Cut, paste, and save: new approaches to altering specific genes in mice. Cell 56, 145-147 9. Hammer, R. E., Krumlauf, R., Camper, S. A., Brinster, R. L., and Tilghman, S. M. (1987) Diversity of alpha-fetoprotein gene expression in mice generated by a combination of separate enhancer elements. Science 235, 53-58 10. Townes, T. M., and Behringer, R. R. (1990) Human globin locus activation region (LAR): role in temporal control. Trends Genet. 6, 219-223 A., Winter,
11. Stief,
D. M.,
Stratling,
W. H.,
and
Sippel,
A. E.
(1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature (London) 341, 343-345 12. Brinster, R. L., Allen, J. M., Behringer, R. R., Gelinas, R. E., and Palmiter, R. D. (1988) Introns increase transcriptional efficiency in transgenic mice. Proc. NaiL Acad. Sci. USA 85, 836-840
13. Palmiter, R. D., Sandgren, E. D. D., and Brinster, R. L. (1991) hance expression of transgenes USA 88, 478-482 14. Rossant, J., and Joyner, A. L. genetic
analysis
of mammalian
P., Avarbock, M. R., Allen, Heterologous introns can enin mice. Proc. Nati. Aced. Sci. (1989) Towards development.
a molecularTrends Genet. 5,
27 7-283 15. Shawlot, W., Siciliano, M. J., Stallings, R. L., and Overbeek, P. A. (1989) Insertional inactivation of the downless gene in a family of transgenic mice. Mol. Biol. Med. 6, 299-307
3000 Vol. 5 November 1991 The FASEB Journal MERLINO ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe
16. Woychik, R. P., Maas, R. L., Zeller, R., Vogt, T. F., and Leder, P. (1990) ‘Formins’: proteins deduced from the alternative transcripts of the limb deformity gene. Nature (London) 346, 850-853 17. Gordon, J. W., Uehlinger, J., Dayani, N., Talansky, B. E., Gordon, M.,
Rudomen,
G. S., and
Neumann,
P. E. (1990)
Analysis
of the
hotfoot (ho) locus by creation of an insertional mutation in a transgenic mouse. Dev. Biol. 137, 349-358 18. Kuehn, M. R., Bradley, A., Robertson, E. J., and Evans, M. J. (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice. Nature (London) 326, 295-298 19. Hooper, M. L., Hardy, K., Handyside, A., Hunter, S., and Monk, M. (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature (London) 326, B. H., Marrack, P., Kappler, J. W., and Smithies, 0. (1990) Normal development of mice deficient in 2M, MHC class I proteins, and CD8 T cells. &ience 248, 1227-1230 21. Zijlstra, M., Bix, M., Simister, N. E., Loring,J. M., Raulet, D. H.,
R. (1990) 32-Microglobulin
deficient
24. 25. 26.
oncogene is required for development of a large region of the mouse brain. Cell 62, 1073-1085 Thomas, K. R., and Capecchi, M. R. (1990) Targeted disruption of the murine mt-i proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature (London) 346, 847-850 Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64, 693-702 Chisaka, 0., and Capecchi, M. R. (1991) Regionally restricted developmental defects resulting from targeted disruption of the mouse homebox gene hox-1.5. Nature (London) 350, 473-479 Stacey, A., Bateman, J., Choi, T, Mascara, T., Cole, W., and Jaenisch, R. (1988) Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineering gene. Nature (London) 332, 131-136
27.
mutant
pro-al(I)
collagen
Ryan, T. M., Townes, T M., Reilly, M. P., Asakura, T., Palmiter, R. D., Brinster, R. L., and Behringer, R. R. (1990) Human sickle hemoglobin
in transgenic
mice.
Science 247,
566-568
28. Greaves, D. R., Fraser, P., Vidal, M. A., Hedges, M. J., Ropers, D., Luzzatto, L., and Grosveld, F. (1990) A transgenic mouse model of sickle cell disorder. Nature (London) 343, 183-185 29. Evans, G. A. (1989) Dissecting mouse development with toxigenics. Genes Dcv. 3, 259-263 30. Readhead, C., Popko, B., Takahashi, N., Shine, H. D., Saavedra, R. A., Sidman, R. L., and Hood, L. (1987) Expression of a myelin basic
protein
gene
in transgenic
shiverer
mice:
correction
of the
dysmyelinating phenotype. Cell 48, 703-712 31. Costantini, F., Chada, K., and Magram, J. (1986) Correction of murine $-thalassemia by gene transfer into the germ line. &ience 233, 1192-1194 32. Mason, A. J., Pitts, S. L., Nikolics, K., Szonyi, E., Wilcox, J. N., Seeburg, P. H., and Stewart, T A. (1986) The hypogonadal mouse: reproductive functions restored by gene therapy. Science 234, 1372-1378
lymphoid transgenic
hyperplasia to high-grade malignant lymphoma in mice for the t(14;18). Nature (London) 349, 254-256 40. Heisterkamp, N., Jenster, G., ten Hoeve, J., Zovich, D., Pattengale, P. K., and Groffen, J. (1990) Acute leukemia in bcr/abl transgenic mice. Nature (London) 344, 251-253 41. Pattengale, P. K., Stewart, T. A., Leder, A., Sinn, E., Muller, W., Tepler, I., Schmidt, E., and Leder, P. (1989) Animal models of human disease: pathology and molecular biology of spontaneous ne-
oplasms occurring vated
in transgenic mice carrying and expressing actiAm. j Pathol. 135, 39-61 E. P., Quaife, C. J., Pinkert, C. A., Palm iter, R. D., and
cellular
Brinster,
oncogenes.
R. L. (1989)
Oncogene-induced
liver neoplasia
in trans-
genic mice. Oncogene 4, 715-724 43. Jhappan, C., Stahle, C., Harkins, R. N., Fausto, N., Smith, G. H., and Merlino, G. T. (1990) TGFx overexpression in transgenic mice
mice lack
CD48 cytolytic T cells. Nature (London) 344, 742-746 22. McMahon, A. P., and Bradley, A. (1990) The Wnt-1 (mt-i) proto-
23.
M. E., Cochrane, K. L., and Field, L. J. (1990) in transgenic mice expressing atrial natriuretic factor fusion genes. Hypertencion 16, 301-307 39. McDonnell, T. J., and Korsmeyer, S. J. (1991) Progression from
42. Sandgren,
292-295
20. Koller,
and Jaenisch,
38. Steinhelper, Hypotension
induces liver neoplasia and abnormal development mary gland and pancreas. Cell 61, 1137-1146
of the mam-
44. Sandgren, E. P., Luetteke, N. C., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1990) Overexpression of 1tFa in transgenic mice: induction
of epithelial
hyperplasia,
pancreatic
metaplasia,
and car-
cinoma of the breast. Cell 61, 1121-1135 45. Matsui, Y., Halter, S. A., Holt,J. T., Hogan, B. L. M., and Coffey, R. J. (1990) Development of mammary hyperplasia and neoplasia in MMTV-TGFa transgenic mice. Cell 61, 1147-1155 46. Vassar, R., and Fuchs, E. (1991) Transgenic mice provide new insights into the role of TGFcs during epidermal development and differentiation. Genes Dcv. .5, 714-727 47.
Silversides, D. W., Oosterloo, M. -L., Merin, L. M., Derynck, R., Ullrich, A., and Overbeek, P. A. (1991) TGFs and EGF transgenes alter developmental morphogenesis of the mouse eye. Development In
press 48. Edwards, R. H., Rutter, W. J., and Hanahan, D. (1989) Directed expression of NGF to pancreatic cells in transgenic mice leads to selective hyperinnervation of the islets. Cell 58, 161-170 49. Tepper, R. I., Levinson, D. A., Stanger, B. Z., Campos-Torres, J., Abbas, A. K., and Leder, P. (1990) IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice. Cell 62, 457-467 50. Dent, L. A., Strath, M., Mellor, A. L., and Sanderson, C. J. (1990) Eisoinophilia in transgenic mice expressing interleukin 5. j Exp. Med. 172, 1425-1431 51. Tominaga, A., Takaki, S., Koyama, N., Katoh, S., Matsumoto, R., Migita, M., Hitoshi, Y., Hosoya, Y., Yamauchi, S., Kanai, Y., Miyazaki, J. -I., Usuku, G., Yamamura, K. -I., and Takatsu, K. (1991) Transgenic mice expressing a B cell growth and differentiation factor gene (interleukin 5) develop eosinophilia and autoantibody production. j Exp. Med. 173, 429-437 52. Lang, R. A., Metcalf, D., Cuthbertson, R. A., Lyons, I., Stanley, E., Kelso, A., Kannourakis, G., Williamson, D. J., Klintworth, G. K., Gonda, T J., and Dunn, A. R. (1987) Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage. Cell. 51, 675-686 53. Galski, H., Sullivan, M., Willingham, M. C., Chin, K. -V., Gottesman, M. M., Pastan, I., and Merlino, G. T. (1989) Expression of a human multidrug resistance eDNA (MDR 1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia. Mol. CelL Biol. 9, 4357-4363 54. Mickisch, G. H., Merlino, G. T., Galski, H., Gottesman, M. M., and Pastan, I. (1991) Transgenic mice that express the multidrugresistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance. Proc. NaiL Aced. Sci. USA 88,
33. Thompson, S., Clarke, A. R., Pow, A. M., Hooper, M. L., and Melton, D. W. (1989) Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56, 313-321 34. Vogel, J., Hinrichs, S. H., Reynolds, R. K., Luciw, P. A., and Jay, G. (1988) The HIV tat gene induces dermal lesions resembling Kaposi’s sarcoma in traisgenic mice. Nature (London) 335, 606-611 35. Leonard, J. M., Abramczuk, J. W., Pezen, D. S., Rutledge, R., Belcher, J. H., Hakim, F., Shearer, G., Lamperth, L., Travis, W., Fredrickson, T., Notkins, A. L., and Martin, M. A. (1988) Develop547-551 ment of disease and virus recovery in transgenic mice containing 55. Isola, L. M., and Gordon, J. W. (1989) Expression of a methotrexHIV proviral DNA. Science 242, 1665-1670 ate resistant dihydrofolate reductase gene in transgenic mice. Dcv. 36. Mullins, J. J., Peters, J., and Ganten, D. (1990) Fulminant hyperGen. 10, 349-355 tension in transgenic rats harbouring the mouse Ren-2 gene. Nature 56. Westphal, H. (1989) Transgenic mammals and biotechnology. (London) 344, 541-544 FASEBJ 3, 117-120 37. Ohkubo, H., Kawakami, H., Kakehi, Y., Takumi, T, Arai, H., L. (1990) The mammary gland as a bioreactor: Yokota, Y., Iwai, M., Tanabe, Y., Masu, M., Hata, J., Iwao, H., 57. Hennighausen, production of foreign proteins in milk. Prot. Exp. Purzf 1, 3-8 Okamoto, H., Yokoyama, M., Nomura, T., Katsuki, M., and Nakanishi, S. (1990) Generation of transgenic mice with elevated 58. Pursel, V. G., Pinkert, C. A., Miller, K. F., Bolt, D. J., Campbell, blood pressure by introduction of the rat renin and angiotensinogen R. G., Palmiter, R. D., Brinster, R. L., and Hammer, R. E. (1989) genes. Proc. NatL Acad. Sci. USA 87, 5153-5157 Genetic engineering of livestock. Science 244, 1281-1288
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