The use of transgenic animals to study the role of growth factors in endocrinology

16 The use of transgenic animals to study the role of growth factors in endocrinology R. F. SEAMARK

INTRODUCTION It is aximotic that a healthy democratic state is dependent on a continuing exchange of information between people. This book reflects the growing recognition that the axiom applies equally to the democracy of cells constituting the animal organism. Due in part to the limitation of available technology, classical endocrinologists, like classical historians, perceived all systems as being organized hierarchically and assumed that they could be comprehended in terms of master (glands) and subservients (target organs and tissues). The new level of focus achievable through modern analytical techniques directly challenges this oversimplistic view. The sophistication of cell-eell communication through the medium of growth factors and the like, the subtlety of the system in achieving cooperation through the co-ordinated action of endocrine and central nervous systems and the challenge these new perspectives present to the scientist attempting to comprehend such an organism is now clearly revealed. This chapter attempts to identify the special role of transgenesis in elucidating the role of the growth factors in the cell-eell communication process.

TRANSGENESIS, THE TECHNIQUE IN PERSPECTIVE The term 'transgenesis' was coined by Gordon and Ruddle (1983) to describe a technical process enabling transfer of inheritable functional genes between organisms irrespective of species barriers. Recent usage has extended its meaning to include not only gene transfer, but manipulation of resident genes to allow over- and underexpression in designated tissues at different stages of development. Simply stated, transgenesis is a technology which allows molecular 'tinkering' in the whole animal. Thus, through transgenesis, the endocrinologist will be able to apply 'ablation and replacement', the classical experimental strategy used to study Bailliere's Clinical Endocrinology and Metabolism833 Vol. 5, No.4. December 1991 Copyright© 1991, by BailliereTindall ISBND-702D-1491-5 All rights of reproduction in any form reserved

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endocrine function in whole animals and to found endocrinology as a science, at the cellular and molecular level. Transgenesis is therefore of special interest in the study of growth factors, which mostly act locally in an autocrine or paracrine manner and are typically multicellular in origin , features which make them particularly difficult to characterize using classical experimental approaches developed to investigate the hormones originating in specialized cells in discrete glands. Most importantly, transgenesis offers new opportunities for the study of the cellular and the genetic and epigenetic determinants of growth factor production and their target cell response during embryonic development and fetal developmental stages, hitherto largely inaccessible to the endocrinologist. Microinjection Transgenic mice are now created as a matter of routine in many laboratories throughout the world, mainly using microinjection, although procedures based on viral vectors and embryonic stem (ES) cells are attracting increasing attention. The microinjection procedure, first developed by Gordon et al (1980), involves introducing the DNA of interest via fine glass pipettes directly into the pronucleus of a recently fertilized egg. The use of the one-cell zygote ensures that, if integrated in the genome, the introduced DNA or transgene will occur in every cell in the body, including the germ cells, and thus be inheritable. Usually, the transgene can be successfully incorporated into about 1-5% of eggs by this procedure, most commonly as concatamers in a head-to-tail array inserted at a single locus. These insertions are inherited as autosomal dominants (Palmiter and Brinster, 1986). The site of insertion is not controlled and can occur anywhere within the genome, with occasional unsolicited consequences (Mahon et al, 1988; Constantini et al, 1989). However, study of the expression of transgenes in their various unique loci has had the major benefit of providing considerable insight into the relative importance of genetic and cellular elements determining gene function. The result is that sufficient is now known about such determinants that it is feasible to include promoter-enhancer elements in the transgene to provide a measure of control of the tissue site and timing of gene expression, largely independent of integration site. Microinjection is the most commonly used technique for inserting genes into the mammalian germ line and has emerged as the method of choice because, of all the physical and chemical techniques currently used in molecular biology laboratories to transfer genes to cells, it is the only one efficient enough for use with oocytes. Most gene transfer procedures are developed for use with cultures of somatic cells with less than 1 in 106 to 105 cells being transformed (Celis, 1984). This inefficiency is tolerated because there is generally an abundance of cells available and it is possible to include selection procedures in the protocol to allow isolation and recovery by cloning of successfully transformed cells.

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Viral vectors Viral vectors, particularly those based on the highly infectious retroviruses, present a viable option for some transgenes (Wagner, 1985; Stewart et ai, 1987) but the size of the DNA that can be inserted through this route is limited, and, whilst the virus vector ensures efficient incorporation without major disruption to the genome, the site of insertion is unpredictable. Difficulties have also been experienced in actually controlling the expression of transgenes introduced by this route (Gordon, 1989). ES cells The emerging transgenesis procedures of choice are those based on ES cells (Robertson, 1986). These offer considerable potential, as they not only promise to overcome many of the problems inherent in gene transfer through microinjection and viral vectors but they allow a new range of possibilities. Typically, ES cells are pluri-, if not toti-potent cells derived from the inner cell mass of blastocyst stage embryos and are maintained in vitro under conditions which allow their continuous proliferation without differentiation. Such cell lines are amenable to genetic manipulation, utilizing the full range of recombinant DNA procedures used in somatic cell research, including site-directed insertion of transgenes and manipulation (mutation) of resident genes in situ, made possible through homologous recombination (Baribault and Kemler, 1989; Capecchi, 1989a,b). The individual ES cells with the targeted transformants can then be selected and cloned and reinstated within a host embryo to contribute to the germ line, using established micromanipulation procedures. At present, successful creation of transgenic animals by the ES route is restricted to a limited number of breeds of mice but there are now reports of ES cell lines being established from hamster (Doetschmann et ai, 1988), pig and sheep (R. M. Moor, personal communication) embryos, and rapid progress may be anticipated in developing ES cells of other species, following the identification of differentiation inhibiting factors hitherto derived through co-culturing the ES cells with specific cell lines secreting the factors (Williams et ai, 1989).

TRANSGENESIS IN GROWTH FACTOR RESEARCH As may be judged from the preceding chapters in this book, growth factor research is undergoing an explosive expansion and it would be impossible to adequately cover all potential applications of transgenesis to this field. Furthermore, the increasing recognition of the role of transgenesis in biological, medical and agricultural research ensures the topic is subject to constant authoritative review (see Palmiter and Brinster, 1986; Gordon, 1989), identifying both the potential and limitations of the technology. Consequently, material selected for this brief chapter will be illustrative

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only and confined to examples which best exemplify the potential of the technology to growth factor research. Creation of models of endocrine dysfunction

Hyperfunctlon The demonstration in 1982 of the dramatical1y enhanced growth of transgenic mice expressing heterologous somatotropin genes under the control of a murine metallothionein (MT) promoter-enhancer sequence captured both the scientific and popular imagination (Palmiter et aI, 1982, 1983). Prior to these experiments, the lifetime effects of continuous ectopic production of hormones could only be inferred through clinical studies or from data obtained from short-term infusion experiments using the larger laboratory animals. Through transgenesis, overexpression of specific growth factors can be achieved in a variety of ways. In the 'supermouse' experiments the cellular site of expression was directed to the large organs (liver, kidney) and expression enhanced through including metal-inducible control elements in the transgene and exposing the animals to zinc. This approach, aimed at achieving large-organ specificity and transgene expression that can be regulated, has been widely imitated. The murine MT-I promoter has proved very useful for this purpose, but heterologous promoters such as the sheep MT-IA promoter, which is Zn-inducible but with intrinsic low basal level activity (Peterson and Mercer, 1986), and modifications of the wellcharacterized human MT-IIA promoter (Karin and Richards, 1982), arc proving more reliable. As heterologous promoters including those from prokaryotic sources can function in the mouse, there is an almost infinite range of possibilities for achieving the requisite control of transgene expression (see Seamark and Wells (1990) for a recent review). This potential wi11 increasingly be realized as information is accumulated concerning the exact mechanism of action of cis- and trans-acting factors and influence of insertion site. An alternative approach used to generate useful animal models has targeted expression of oncogenes to cause hyperplasia of the endocrine tissue (Ornitz ct aI, 1987). This approach has the added advantage that in some instances the hyperplastic tissues can provide a source of transformed, immortalized cell lines (Cory and Adams, 1988). The now numerous transgenic animals created with novel endocrine capacity arc providing valuable insight into the endocrine systems under study. For example, whilst many of the physiological and phenotypic consequences of chronic overexpression of growth hormone (GH) in transgenic mice, such as the excessive growth and profound atrophy of pituitary acidophilic cells, were predictable from previous animal experiments, others such as the associated sex-specific alteration in hepatic function, the reduced fertility of the female transgenic mice and the disturbances of liver and kidney function were largely unexpected, and arc now the subjects of active research (Brem et al, 1989; Quaife et al, 1989).

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Transgenesis can also be used as a means of exposing animals to growth and other factors at stages of their development when they would not usually be accessible to scientific manipulation. The fetal 'supermice', for example, were exposed to high levels of GH (Palmiter et ai, 1982). This has exciting implications in embryological and fetal research. Hypofunction Cellular ablation

Transgenesis provides a unique means of targeting and ablating cells producing a specific growth factor. For example, in studies of growth regulation, confirmation of the critical role of the somatotrope was achieved by cellular ablation (Palmiter et ai, 1987). This was carried out by creating transgenes comprising control sequences obtained from the (rat) GH gene, to achieve tissue specificity, fused to structural genes producing a cytotoxin (diphtheria toxin). Later, an enzyme (thymidine kinase, TK; Borrelli et ai, 1988) was used for the same purpose which caused the target cell (somatotrope) to acquire pharmacological sensitivity to synthetic nucleosides whose metabolites kill dividing cells. This latter approach has the benefit that the cytotoxicity is inducible, thus avoiding potentially lethal effects of premature expression during embryo or fetal stages. A high level of cellular specificity is potentially achievable by this means but there are limitations. In the example cited above, for instance, the stated objective of producing transgenic mice lacking the somatotropes exclusively was not achieved. The transgenic mice produced lacked somatotropes and were dwarfed but because the somatotrope shares a common cellular origin with the lactotrope, the pituitaries of the transgenic animals lacked both cell types (Palmiter et ai, 1987; Borrelli et ai, 1988). Interestingly, in a concomitant attempt to specifically ablate the lactotrope, using the same regulatable TK obliteration system but fused with a promoter sequence from the rat prolactin gene (Borrelli et ai, 1988), transgenic mice were created which expressed TK in the lactotropes but which retained anatomically and histologically normal pituitaries, following exposure to the synthetic nucleotide. Furthermore, the putative common stem cells of the lactotrope and somatotrope were apparently still present in the adult animals as they proved capable of repopulating the pituitary of the nucleoside-treated animals. Molecular ablation

Cell ablation achieved through the transgenesis approaches described, particularly the inductive (TK) type, has resulted in many useful research models; however, as stated, disruption occurs at the cellular and not the molecular level. With the development of ES cell technology to a routine, further refinement will be possible, including disruption of the specific growth factor genes. The harbinger of such development is to be found in the

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recent report of successful ablation of the insulin-like growth factor II (IGF-II) gene in mice (De Chira et aI, 1990). The resultant growthdeficiency phenotope provides the first direct confirmation of the growthpromoting role of this factor in fetal development. ES cell technology is, however, not as yet routine. Reversible regulation at the molecular level may be achieved through the conventional microinjection approach, using transgenes containing antisense sequences (see van der Krol et al (1988) for a recent review). These transgenes are designed to contain appropriate promoter sequences to ensure effective expression in the specific tissue cells of interest, and a DNA construct which generates RNA complementary to the mRNA of the growth factor of interest. This approach, although it has not as yet been widely applied to growth factor studies, is already proving to be an extremely powerful tool in the manipulation of embryogenesis. Alternative approaches

Immune modulation Many endocrine disorders are now recognized as being consequential on disturbances in normal immune surveillance and there have been several attempts to mimic these disturbances through transgenesis (see Cuthbertson and Klintworth, 1988). For example, overexpression of a class II MIIC structural gene linked to the insulin promoter was found to lead to pancreas B cell depletion (Lo et ai, 1988; Sarvetnick et al, 1988), and this was cited as a potential model of insulin-dependent diabetes mellitus caused by autoimmune destruction of the B cell. However, unexpectedly for an autoimmune disease, B cell destruction occurred in all mice overexpressing the gene (Allison et al, 1988) and, as judged from a subsequent study with neonatally thymectomized and nude mice (Miller et al, 1989), there was no evidence of lymphocyte involvement, leading to the conclusion that overexpression of the class II MHC gene itself provided the cytotoxic factor. All products of transgene expression are potentially immunogenic and class I and II MHC genes can be expressed appropriately in transgenic mice and their expression can alter the immune response of the animals (Adams et al, 1987). Thus, immune-mediated cellular and molecular ablation offers many interesting prospects. However, whether specific gene products are recognized as such is dependent on the stage of development at the time of initial expression and this may prove a significant constraint to the broader usage of this approach.

Insertional mutations The present lack of control of the site of gene insertion raises the constant prospect of causing insertional mutations. This has occasionally had useful results, such as the valuable animal model of the growth hormone-resistant human dwarf syndrome, which was generated recently by this means (Xiang et aI, 1990). This rare, autosomal recessive mutant line was found to harbour

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two different integration patterns of a transgene at the same locus, the locus proving aJleleic to a spontaneous mutant pygmy (Xiang et al, 1990). Whilst the recognition of useful mutants is demanding, a number of transgenic insertional mutants have been isolated by this approach and characterized at the molecular level (see Xiang et al (1990) for references). and there is growing recognition of the potential of utilizing transgenes as insertional mutagens and then as probes to clone the disrupted loci (Allen et al, 1988; Gossler et ai, 1989). Serendipity Several valuable transgenic models of endocrine dysfunction have been developed fortuitously. For example, several lines of transgenic mice were generated exhibiting ectopic expression of the human GH transgene localized to the cerebral cortex (Hollingshead et al, 1989) during the course of experiments on the role of the mouse mammary tumour virus promoter. In these experiments the (human) GH gene was simply present as a reporter sequence. Presumably the viral sequences acted to mimic cortex-specific trans-acting factors. Other useful models have emerged from studies involving viral sequences which develop cis-acting elements with unusual tissue specificity when combined with certain structural genes or promoter sequences (Palmiter et al, 1985).

INVESTIGATION OF GROWTH FACTOR PRODUCTION

Regulatory factors Transgenesis clearly has a special role to play in the investigations of the nature of factors regulating gene expression in vivo (Jami, 1988), and will be a method of choice in ascribing the relative importance to cis- and transacting factors and of the integration site in the differential regulation of specific growth factor genes. As was quickly appreciated from the early transgenic mouse experiments, cis-acting factors have proven to be the major determinants of tissue specificity, with the characteristic level of expression determined not only by the strength of the enhancer but accessibility to trans-acting factors (see review by Gordon, 1989). The latter finding was unexpected as impressions gained from studies of tissue culture cells were that individual enhancers tended to have equivalent strength. The location within the genome of the cis-acting elements relative to the structural gene confirming tissue specificity varies. For example, with the elastase gene the important region conferring pancreas specificity expression occurs within 200 base pairs of DNA upstream (5') to the gene (Davis and MacDonald, 1988). By contrast, sequences up to 6500 base pairs 5' to the gene are involved in

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tissue-specific expression of the o-fetoprotein gene (Godbout et aI, 1986). Other variations include controlling elements within intron sequences as exemplified by the immunoglobulin heavy chain gene enhancer (Banerji et aI, 1983) and tissue-specific regulatory elements being found with both 5'· and 3'-coding regions as with globin genes. Transfer of putative cis-acting sequences from, say, a growth factor of interest fused with appropriate reporter sequences to mice can be used to provide valuable insight into both genetic and epi-genetic factors regulating gene expression (Jami, 1988; Portanova et aI, 1990). As stated previously, cis-acting elements appear as being major regulatory factors and to express appropriately in mice, even when derived from heterologous species. Transacting factors appear to be available in mouse tissue to generally ensure a pattern of expression indistinguishable from that found in the promoter sequence donor gene. However, differences can exist during early developmental stages as the trans-acting factors are themselves gene products and individual species can differ in the developmental stage when gene expression is initiated (Krumlauf et al, 1985). Mice have an inherent advantage over other mammalian experimental animals in that many inbred strains and interstain hybrids are available, making it possible to study gene regulation using classical genetics. The resolution of the genetic basis of the pleiotrophic activity of trans-actory factor which influences a fetoprotein transcription is an example of this (Pandris et aI, 1984). Recently, the use of transgenesis has been extended to the study of transcriptional activity of individual nuclear factors (Verwey et aI, 1990), indicating its potential in studies of intracrine regulatory mechanisms in growth factor activity (Logan, 1990).

Ontogeny of gene expression Transgenesis has a special role in studies of the ontogeny of gene expression and has a major part to play in studies concerned with growth factors in the early developmental stages. Through ingenious use of heterologous genes or reporter sequences attached to presumptive cis-acting factors, detailed information on the developmental genetics is now available for a wide range of systems (Gordon, 1989). Novel uses of transgenesis are also emerging, such as the use of transgenes as probes for active chromosomal domains during mouse development (Allen et aI, 1988). This introduces a completely new range of prospects for identifying endogenous genes involved in organogenesis and pattern formation. Importantly, in these and all studies based on this technology, transgenesis retains a capacity to surprise. This is exemplified by the recent unexpected finding that transgenic female mice overexpressing Mullerian inhibitory substance (MIS), a sex-differentiating factor normally produced by the fetal testes, lacked ovaries (Behringer et aI, 1990). Several of the males also exhibited an unusual phenotype with feminized external genitalia, abnormal Wolffian duct development and undescended testes. Such unforeseen sequelae provide essential stimuli to research.

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INVESTIGATION OF GROWTH FACTOR ACTION Target cells The range of laboratory procedures now available to identify, isolate and characterize hormone receptor genes ensures their increasing availability for transgenesis-based investigations. Experiments aiming to establish new or enhance existing sensitivity of target tissues to growth factors through overexpression of receptor genes (Vonboehmer, 1990; Patil et ai, 1990) are well advanced. If appropriate, transgenesis could be utilized to specifically ablate target cells (see earlier) and modify receptor gene expression (see earlier). Through utilizing ES cell technology (Baribault et ai, 1989; Capecchi, 1989a,b), receptor specificity and function could also be altered. All these possible approaches may be needed to dissect the network of interaction between various growth factors, and the target cell matrix. As is clearly revealed in the present volume, appreciation of the complexity of these interactions is growing rapidly and there is a developing need for new approaches, such as transgenesis, which will allow experimentation in a more physiological context. Modulatory factors Again, as emphasized in other chapters of this book, control of growth factor function may be determined at many levels. It has long been known that the biological efficacy of circulating hormones can be influenced to a greater or lesser degree by binding proteins present in the plasma, and recent studies indicate that they play an important part in determining the biological activity of paracrine and autocrine factors. The growth-promoting activity of IGF-I, for example, is known to be influenced by the presence of at least five binding proteins (Sara and Hall, 1990). How much these binding proteins were able to influence IGF-I activity was recently indicated by the discovery of a truncated IGF-I variant which, though lacking three N-terminal amino acids, escapes binding. In the absence of binding, this variant is proving to be up to five times more active as a cell growth promoter than IGF-I itself (Ballard et al, 1989). Manipulation of the hormone-binding proteins of the growth factors themselves through transgenesis would allow new possibilities in quantifying the physiology significance of such modulatory influences. This would be of special interest in differing physiological situations such as pregnancy, where there is an enhanced output of specific binding proteins in uterine tissue and plasma (Bell et al, 1988). Investigations of endocrine systems The role of transgenes in providing animal models which challenge our understanding of how endocrine systems arc organized is clearly exemplified by the outstanding series of studies on growth regulation initiated by Ralph Brinster and Richard Palmiter and their associates. In addition to the

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variants already referred to, mice transgenic with growth hormone releasing factor (GRF) (Frohman et aI, 1989), somatostatin (Low et aI, 1985) and IGF-I (Mathews et aI, 1988a,b) genes have been produced and the multiple consequences of their overexpression utilized as probes both to confirm existing and realize new features of the growth system. In addition, new mutants have emerged which are providing useful models of human genetic disease (Asa et aI, 1990) and the potential raised of correcting such inherited defects (Manson et aI, 1986; Palmiter and Brinster, 1986). The new dimension of creating recessive mutants through gene deletion is already impacting on studies of growth factors such as IGF-II (De Chiara et aI, 1990), and it may be confidently predicted that research into all endocrine systems will benefit from this technology. Transgenesis is already providing provocative input into research on the complex system of cytokines which regulate the haemopoietic and lymphoid systems (Storb, 1987; Cory and Adams, 1988), and its use is rapidly extending to the renin-angiotensin (Clouston et aI, 1989; Digby et aI, 1989; Patil et aI, 1990), liporegulatory (Hofmann et aI, 1988) and reproductive systems (Chandrashekar et aI, 1988; Behringer et aI, 1990). Transgenic animals can also act as sources of growth factors for clinical investigational research and treatment (Costa et aI, 1990).

SUMMARY Transgenesis is identified as being of special interest in the study of growth factors where their multicellular origins and complex interactions make them particularly difficult to characterize using classical experimental approaches developed to investigate hormones originating in specialized cells in discrete glands. Through allowing molecular 'tinkering' in intact animals, transgenesis enables specific growth factors to be 'ablated or replaced' from specific tissues and organs and target cell response and impact of modulatory factors such as binding proteins to be explored in the intact animal. To the endocrinologist, the potential applications of such technology are legend. This chapter provides a brief overview of the technique and provides linkages to the rapidly developing body of literature in establishing transgenesis in growth factor research. Acknowledgement I wish to thank Glenys King for her patient typing of the manuscript.

REFERENCES Adams TE, Alpert S & Hanahan D (1987) Non-tolerance and autoantibodies to a transgenic self antigen expressed in pancreatic beta cells. Nature 325: 223-228. Allen ND, Cran DG, Barton SC et al (1988) Transgenes as probes for active chromosomal domains in mouse development. Nature 333: 852-855. Allison J, Campbell I, Morahan G et al (1988) Diabetes in transgenic mice resulting from

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over-expression of class I histocompatibility molecules in pancreatic beta cells. Nature 333: 529-533. Asa SL. Kovacs K. Stefaneanu L. Horvath E. Billestrop N & Gonzalcz-Cauchon C & Vale W (1990) Pituitary mammosomatotroph adenomas develop in old mice transgenic for growth hormone releasing factor. Proceedings of the Society of Experimental alld Biological Medicine 193: 232-235. Ballard RJ. Francis GL. Bagley CJ. Szabo L & Wallace JC (1989) Effects of insulin-like growth factors on protein metabolism. Why arc some molecular variants more potent? Biochemical Society Symposia 55: 91-104. Bancrji JT. Olsen L & Schattner W (1983) A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33: 729-740. Baribault H & Kemler R (1989) Embryonic stem cell culture and gene targeting in transgenic mice. Molecular Biology and Medicine 6: 4SI-t92. Behringer RR. Mathews L. Palmiter RD & Brinster RL (1988) Dwarf mice produced by genetic ablation of growth hormone-expressing cells. Genes and Development 2: 453-t61. Behringer RR. Cate RL. Froclick GJ. Palmiter RD & Brinstcr RL (1990) Abnormal sexual development in transgenic mice chronically expressing Mullerian inhibiting substance. Nature 345: 167-170. Bell SC. Pate ISR. Jackson JA & Waites GT (1988) Major secretory protein of human dccidualizcd endometrium in pregnancy is an insulin-like growth factor binding protein. Journal of Endocrinology 118: 317-328. Borrelli E. Heyman R. Hsi M & Evans R (1988) Targeting of an inducible toxic phenotype in animal cells. Proceedings ofthe National Academy of Sciences ofthe USA 85: 7572-7576. Brcrn G. Wanke R. Wolfe E ct al (1989) Multiple consequences of human growth hormone expression in transgenic mice. Molecular and Biological Medicine 6: 531-547. Capccchi MR (1989a) Altering the genome by homologous recombination. Science 2~: 1288-1292. Capecchi MR (1989b) The new mouse genetics: altering the genome by gene targeting. Trends in Generics 5: 7()--{). Celis JE (1984) Microinjection of somatic cells with micropipettes: comparison with other transfer procedures. Biochemistry Journal 223: 281-291. Chandrashekar V, Bartke A & Wagner TE (1988) Endogenous human growth hormone (Gil) modulates the effect of gonadotrophin-releasing hormone on pituitary function and the gonadotrophin response to the negative feedback effect of testosterone in adult male transgenic mice bearing human Gil gene. Endocrinology 123: 2717-2727. Clouston W, Lyons I & Richards R (1989) Tissue-specific and hormonal regulation of angiotensinogen minigenes in transgenic mice. EM BO Journal 8: 3337-3343. Constantini F. Radice G, Lee J, Chada K. Perry W & Son II (1989) Insertional mutations in transgenic mice. Progress in Nucleic Acid Research and Molecular Biology 36: 159-169. Cory S & Adams J (1988) Transgenic mice and oncogenesis. Annual Review of Immunology 6: 25-t8. Costa 1', Sassano M, Righi M & Riccioardicastagnoli P (1990) Transgenic animals as bioreactors for pharmacological products. Pharmacological Research 22: 75-78. Cuthbertson R & Klintworth G (1988) Transgenic mice-a gold mine for furthering knowledge in pathobiology. Laboratory Investigation 58: 48+-502. Davis BP & MacDonald RJ (1988) Limited transcription of rat elastase Itransgene repeats in transgenic mice. Genes and Development 2: 13-22. DeChiara TM, Efstratiadis A & Robertson EJ (1990) A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345: 78-80. Digby M, Lyons I, Fowler K. Homfray M & Richards R (1989) Cell-specific ablation ill transgenic mice-the function of cells expressing kallikrein. Symposium 0/0-00-/; HIe Eleventh Anllual Conference 011 the Organization and Expression of the Genome. 20-24 February, Lome, Victoria. Doctschrnann T, Williams P.& Maeda N (1988) Establishment of hamster blastocyst-derived embryonic stem (ES) cells. Developmental Biology 127: 22+-227. Frohman LA, Downs TR, Chornczynski, Brar A & Kashio Y (1989) Regulation of growth hormone-releasing hormone gene expression and biosynthesis. Yale Journal of Biological Medicine 62: 427-t33.

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Godbout R, Ingram R & Tilgham SM (1986) Multiple regulatory elements in the intcrgenic region between the a-feto protein and albumin genes. Molecular and Cell Biology 6: 477-487. Gordon JW (1989) Transgenic animals. International Revue of Cytology 115: 171-229. Gordon JW & Ruddle FII (1983) Gene transfer into mouse embryos: production of transgenic mice by pronuclear injection. Methods in Enzymology 101: 411-433. Gordon JW, Scangos GA, Plotkin D, Barbosa JA & Ruddle HI (1980) Genetic transformation of mouse embryos by mieroinjeetion of purified DNA. Proceedings uf the National Academy of Sciences of the USA 77: 7380-7384. Gossler A, Joyner AL, Rossant J & Skarnes WC (1989) Mouse embryonic stem cells and reporter constructs to detect developmentally related genes. Science 244: 463-468. Hollingshead PG, Martin L, Pitts SL & Stewart TA (1989) A dominant phenocopy of hypopituitarism in transgenic mice resulting from central nervous system synthesis of human growth hormone. Endocrinology 125: 556--564. Hofmann S, Russel D, Brown 11.1, Goldstein J & Hammer R (1988) Overexpression of low density lipoprotein (LDL) receptor eliminates LDL from plasma of transgenic mice. Science 239: 1277-1281. Jami J (1988) Transgenic mice: a tool for the study of tissue specific gene expression. Nouvelle Revue Francoise Hematologic 30: 7-11. Karin M & Richards RI (1982) Human metallothionein genes-primary structure of the metallothionein-I1 gene and a related processed gene. Nature, London 299: 797-802. Krumlauf RR, Hammer RE, Tilghman SM & Brinster RL (1985) Developmental regulation of alpha-fetoprotein genes in transgenic mice. Molecular and Cellular Biology 5: 1639-1648. Logan A (1990) Intracrinc regulation at the nucleus-a further mechanism of growth factor activity? Iournal of Endocrinology 125: 339-343. Lo D, Burkly LC, Widcra G et al (1988) Diabetes and tolerance in transgenic mice expression class II MIIC molecules in pancreatic beta cells, Cell 53: 159-168, Low MJ, Hammer RE, Goodman RII et al (1985) Tissue-specific post-translational processing of pre-prosornatostatin encoded by a rnethallothioncin-somatostatin fusion gene in transgenic mice, Cell Al: 211-219. Mahon KA, Overbeck PA & Westphal H (1988) Prenatal lethality in a transgenic mouse line is the result of a chromosomal translocation. Proceedings uf the National Academy of Sciences of the USA 85: 1165-1168, Manson AJ, Pitts SL, Nikolies K et al (1986) The hypogonadal mouse: reproductive functions restored by gene therapy. Science 23...: 1372-1378. Mathews LS, Hammer RE, Behringer RR et al (1988a) Growth enhancement of transgenic mice expression human insulin-like growth factor I. Endocrinology 123: 2827-2833. Mathews LS, Hammer RE, Brinster RL & Palmiter RD (l988b) Expression of insulin-like growth factor I in transgenic mice with elevated levels of growth hormone is correlated with growth. Endocrinology 123: 433-437. Miller J. Morahan G, Allison J, Bhathal P & Cox K (1989) T-cell tolerance in transgenic mice expressing major histocompatibility class I molecules in defined tissues, Immunology Review 107: 109-123. Ornitz D. Hammer RE, Messing A, Palmiter RD & Brinster RL (1987) Pancreatic neoplasia induced by SV40 T-antigen expression in acinar cells of transgenic mice. Science 238: 188--193. Palmiter RD & Brinster RL (1986) Germline transformation of mice. AlIlIlIal Review of Genetics 20: 465-499. Palmiter RD, Brinster RL, Hammer RE et al (198:!) Dramatic growth of mice that develop from eggs microinjcctcd with metallothionein-growth hormone fusion genes. Nature 300: 611-615. Palmiter RD, Norstedt G, Gelinas R, Hammer RE & Brinster RL (1983) Mctallothioncinhuman Gil fusion genes stimulate growth of mice. Science 222: 809-814. Palmiter RD, Chen IIY, Messing A &Brinster RL (1985) SV40 enhancer and large T-cell antigen arc instrumental in development of choroid plexus tumours in transgenic mice, Nature 316: 457-463. Palmiter RD, Behringer RL, Quaife C et al (1987) Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell 50: 435-443. Pandris V, Belayew A & Tilghman S1\1 (1984) Locus unlinked to a-Ierroprotein under the

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control of the murine raf and Rif genes. Proceedings ofthe National Academy ofSciences of the USA 81: 5523-5527. Patil N, Lacy E & Chao MV (1990) Specific neuronal expression of human NGF receptors in the basal forebrain and cerebellum of transgenic mice. Neuron 4: 437-447. Peterson MG & Mercer JFB (1986) Structure and regulation of the sheep mctallothioncin-Ia gene. European Journal of Biochemistry 160: 579-585. Portanova R, Yun JS & Wagner TE (1990) Stress-induced secretion of human growth hormone in transgenic mice. Proceedings of the Society of Experimental and Biological Medicine 193: 46-49. Quaife CJ, Mathews LS, Pinkert CA et al (1989) Histopathology associated with elevated levels of growth hormone and insulin-like growth factor-I in transgenic mice. Endocrinology 124: 40-48. Robertson EJ (1986) Pluripotent stem cell lines as a route into the mouse germ line. Trends in Genetics 2: 9-13. Sara VR & Hall K (1990) Insulin-like growth factors or their binding proteins. Physiology Reviews 70: 591-614. Sarvetnick N, Liggitt D, Pitts SL, Hansen SE & Stewart TA (1988) Insulin-dependent diabetes mellitus induced in transgenic mice by ectopic expression of class II MI IC and interferongamma. Cel/52: 773-782. Seamark RF & Wells JRE (1990) Biotechnology and reproduction. Elsevier \Vorld Animal Science Series 14: (in press). Stewart CL, Schuetz S, Vanek K & Wagner ER (1987) Expression of rctroviral vectors in transgenic mice obtained by embryo injection. EMBO Journal in 383-388. Storb V (1987) Transgenic mice with immunoglobulin genes. Annual Review ofImmunology 5: 151-174. van der Kro1 AR, Mol JNM & Stuitje AR (1988) Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques 6: 958-976. Verwey CL, Guidos C & Crabtree GR (1990) Cell type specificity and activation requirements for N fat-I (Nuclear factor of activated T-cells) transcriptional factor activity determined by a new method using transgenic mice to assay transcriptional activity of an individual nuclear factor. Journal of Biological Chemistry 265: 15788-15795. Vonboehmer H (1990) Developmental biology of T-cells in T cell receptor transgenic mice. Annual Review of Immunology 8: 531-556. Wagner EF, Keller G, Gilboa R, Ruther U & Stewart CL (1985) Gene transfer into murine stem cells and mice using retroviral vectors. Cold Spring Harbor Symposia on Quantitative Biology 50: 691-700. Williams RL, Hilton DJ, Pease S et al (1989) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336: 684-687. Xiang X, Benson KF & Chada K (1990) Mini-mouse: disruption of the pigmy locus in a trangenic insertional mutant. Science 247: 967-969.