Cardiac and neurological abnormalities in v-fps transgenic mice

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 5873-5877, August 1989 Genetics

Cardiac and neurological abnormalities in v-fps transgenic mice (protein-tyrosine kinase/cardiomyopathy/oncogene)

SIU-POK YEE*, DAVID MOCKtt, VICTOR MALTBY*, MALCOLM SILVERt, JANET ROSSANT*§, ALAN BERNSTEIN*§, AND TONY PAWSON*§ *Division of Molecular and Developmental Biology, Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto, ON, Departments of §Medical Genetics, tPathology, and tOral Medicine, University of Toronto, Toronto, ON, M5G 1LS, Canada

M5G 1X5 Canada; and

Communicated by Raymond L. Erikson, April 24, 1989 (received for review January 15, 1989)

adult human P-globin promoter was isolated as a 1.6-kb fragment upstream of the initiation codon by digestion with Bgl II and Nco I. The Nco I site was removed with S1 nuclease and replaced with a HindIII linker. The 8-globin promoter fragment was then inserted on the 5' side of the gag-fps coding sequence. For pGF the poly(A) signal was derived from simian virus 40 (SV40) as a Bcl I-EcoRI fragment that was inserted on the 3' side of the gag-fps sequence. The poly(A) signal for pGEF was derived from the 2.7-kb BamHI-Xba I3' fragment of the human ,-globin gene. This fragment was subcloned into pUC19 carrying a Bgl II linker at the HincII site to generate a 3' Bgl II site, removed with BamHI and Bgl II, and cloned downstream of the gag-fps sequence. Plasmid DNAs were digested with Bgl II (pGEF) or Bgl II and EcoRI (pGF). Purified inserts were microinjected into fertilized CD1 mouse eggs (Charles River Breeding Laboratories). All microinjection and oviduct transfer procedures were carried out according to Hogan et al. (16). RNA Isolation and Northern Blot Analysis. RNAs were isolated from tissues essentially by the procedure of Auffray and Rougeon (17). RNA (5-20 pug) was fractionated using formaldehyde denaturing gel electrophoresis. Northern blot analysis was carried out according to Maniatis et al. (18). fps-specific RNA was detected by hybridization with a 2.75kb v-fps BamHI fragment radiolabeled with [a- 32P]dCTP by nick-translation (19). Assays for P130sag-fPs Kinase Activity. Freshly excised hearts were washed with isotonic phosphate-buffered saline (PBS), disrupted in a Polytron (Brinkmann) in RIPA buffer (50 mM TrisHCl, pH 7.5/150 mM NaCl/0.1% SDS/1% sodium deoxycholate/1% Triton X-100/2 mM phenylmethylsulfonyl fluoride/leupeptin (100 t&g/ml)/100 ,tM sodium orthovanadate), and centrifuged for 15 min at 3000 x g to remove insoluble debris. The supernatants were precleared by incubation at 4°C for 1 hr with rabbit anti-mouse immunoglobulin antibody and heat-inactivated formalin-fixed Staphylococcus aureus. S. aureus was removed by centrifugation, and 10 ,u1 of the resulting supernatant was removed from each sample for protein determination using the BCA protein assay (Pierce). Samples, each containing equal amounts of protein, were divided into two aliquots and immunoprecipitated with either R254E anti-pl9gag mouse monoclonal antibody or a control mouse monoclonal antibody. Immune complex kinase reactions to assay for P130'ag4PS autophosphorylation were performed essentially as described (15, 20). Immunoprecipitates were incubated in 10 mM MnCl2/10 mM Hepes, pH 7.0, with 50 ,uCi of [y-32P]ATP (1 Ci = 37 GBq) for 15 min at 25°C, and the kinase reaction products were separated in a SDS/7.5% polyacrylamide gel. The gel was alkalitreated by incubation for 90 min in 1 M KOH at 55°C to enrich for phosphotyrosine, dried, and exposed to x-ray film.

Transgenic mice that widely express the v-frs ABSTRACT protein-tyrosine kinase develop several independent pathological conditions, in addition to a high tumor incidence. v-frs expression and protein-tyrosine kinase activity in the heart were directly correlated with cardiac enlargement. This cardiomegaly was accompanied by severe myocardial and endocardial damage, which was concentrated in the left ventricular wall, and characterized by a progressive atrophy and necrosis of cardiac muscle fibers with concomitant fibrosis. This pathology was associated with congestive heart failure. Mice from five lines developed a marked trembling, correlated with expression of the v-fs transgene in the brain, and two lines showed a striking bilateral enlargement of the trigeminal nerves. Unlike tumor formation, these cardiac and neurological phenotypes were evident shortly after birth and showed 100% penetrance. The pleiotropic effects of the v-fps transgene suggest the involvement of protein-tyrosine kinases in mammalian neural development and cardiac function.

Modification of the mouse germ line by the introduction of foreign genes provides a powerful approach to investigate cellular regulation in both normal and disease states (1-7). A large and diverse family of cellular genes encode transmembrane (receptor-like) or cytoplasmic (nonreceptor) proteintyrosine kinases (PTKs) (8). Transmembrane PlKs have in some instances been identified as receptors for hormones that regulate cellular metabolism, proliferation, or differentiation (9). In general, the physiological activities of cytoplasmic PTKs, such as those encoded by the src, fps/fes, and abl genes, are unclear (8). The expression of some PTKs in post-mitotic cells and in restricted cell lineages has suggested that they regulate the phenotypes and interactions of mature cells, as well as interpreting proliferative and developmental signals in differentiating cells (10-12). To address this issue we introduced the v-fps/fes oncogene, encoding a constitutively active cytoplasmic PTK (13, 14), into the mouse germ line. We wished to achieve widespread expression of the transgene to identify cellular processes that were sensitive to elevated tyrosine kinase activity. We reasoned that the identification of such cells or tissues would illustrate the involvement of endogenous PTKs in normal or abnormal physiology. Here we report that expression of the v-fps PTK in transgenic mice induces a pleiotropic phenotype that includes cardiac and neurological disorders.

MATERIALS AND METHODS Plasmid Constructs and Embryo Microinjection. The gagfps coding sequence was obtained from the pIV2 vector (15) as a 3.8-kilobase (kb) HindIII-Nru I fragment that was modified by ligation of a Bgl II linker to the Nru I site. The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: PTK, protein-tyrosine kinase; SV40, simian virus 40.

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Histopathology. Complete autopsies were performed. Tissues were fixed in PBS-buffered 10% (vol/vol) formalin, embedded in paraffin, sectioned at 7 Am or 3 Am, and stained with hematoxylin and eosin.

Table 1. Phenotypes observed in GF and GEF transgenic mice Line Tremors Cardiomegaly GF2 + GF3 GF6 GEF1 + + GEF3 + + + + GEF4 GEF6 + + Mice from all lines were of a small size and developed tumors with

RESULTS Generation of v-fps Transgenic Mice. Preliminary work with a construct in which v-fps coding sequences were cloned downstream of a human p-actin promoter suggested that extensive v-fps expression might be lethal during embryogenesis, as described for the polyomavirus middle tumor antigen (7). To overcome this toxicity, we placed v-fps coding sequences under the control of weak or tissue-specific transcriptional elements, employing initially human 13-globin regulatory elements. The GF construct contains the human f3-globin promoter placed 5' to the gag-fps sequence of Fujinami sarcoma virus, which encodes a 130-kDa cytoplasmic PTK (P13094Ps) (13, 14). An SV40 polyadenylylation site was cloned 3' of gag-fps to generate the GF minigene (Fig. 1). In an effort to direct gag-fps expression to erythroid cells a second minigene, GEF, was constructed by replacing the SV40 polyadenylylation sequence of GF with a 2.7-kb BamHI-Xba I fragment derived from the 3' end of the human P3-globin gene (Fig. 1). This 3' P3-globin fragment provides a polyadenylylation site and carries cis-acting elements that regulate appropriate erythroid-specific and temporal expression of the human 83-globin locus in transgenic mice (21-23). The GF and GEF minigenes were freed of plasmid sequences and microinjected into mouse embryos to generate transgenic animals. Three founder animals were produced that carried the GF sequence (GF2, GF3, and GF6), and four founders were identified with the GEF transgene (GEF1, GEF3, GEF4, and GEF6). The founders were bred with normal CD1 animals and transmitted the transgenes to their progeny in a Mendelian fashion with the exception of GF3. The GF3 founder was presumed to be mosaic as only 2 out of 48 offspring were transgenic; in each case subsequent inheritance was as predicted for a normal autosomal locus. Southern blot analysis of tail DNA demonstrated multiple copies of the transgene (between 5 and 40 copies depending on the founder) in a linear head-to-tail organization. No obvious rearrangements of the transgene sequences were detected in any of the animals. Contrary to expectation, the GEF as well as the GF constructs were expressed in a wide variety of tissues in all lines and induced a range of specific physiological and neoplastic abnormalities, as summarized in Table 1. v-fps Transcripts Are Widely Distributed in GF and GEF Transgenic Mice. To investigate the expression of the GEF transgene, total RNA was extracted from a variety of tissues of GEF mice and subjected to Northern blot analysis. The probe recognized a 4.2-kb RNA in all GEF lines that was most abundant in heart, followed by brain, thymus, lymph BgIlI

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nodes, testes, and spleen, and barely detectable or absent from lung, blood, salivary gland, kidney, and liver (Fig. 2). Northern blot analysis of v-fps RNA from mice of the three GF lines showed a very similar tissue distribution (data not shown) with the exception of heart, which had little GF RNA, and brain, which only had detectable expression in the GF2 line (see Fig. 5B). Cardiovascular Abnormalities in GEF Mice Are Correlated with v-fps Tyrosine Kinase Activity in the Heart. All mice of lines GEF1, GEF3, GEF4, and GEF6, hemizygous for the GEF transgene, developed a marked reddening of the ears, paws, and tails, that grew increasingly striking with age (data not shown). This phenotype was strictly associated with the GEF transgenic mice and was not seen in animals with the GF transgene. In all four GEF lines penetrance of this trait was 100%. The subcutaneous blood vessels of such animals were enlarged, although the vessels themselves appeared histologically normal. At autopsy these GEF animals were found to have various degrees of cardiac enlargement. This cardiomegaly, which was most pronounced in GEF3 mice, was accompanied by increases in chamber size, in the weights of both ventricles and atria and in the thickness of the myocardium (data not shown). Histopathological examination of hearts from animals of the GEF lines revealed a series of changes characterized by progressive atrophy and necrosis of the cardiac muscle fibers, with replacement of the myofibers with fibrous tissue (Fig. 3). Lesions were first noted as foci of interstitial mononuclear cells surrounding or replacing small groups of cardiac fibers anywhere in the myocardium. More severe myocardial damage was distinguished by larger areas of atrophied or necrotic myofibers concentrated in the subendocardial one-third to one-half of the left ventricular wall, although they could also be found in the subendocardial zone of the right ventricle and elsewhere in the myocardium. Inflammatory cells, especially macrophage-like cells, and proliferating fibroblasts surrounded the necrotic areas. Advanced lesions showed the inner one-third to one-half of the left ventricular wall partially or completely replaced by cellular fibrous tissue, with organized mural thrombi en-

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and most dramatic and constant in mice of the GEF3 lineage (Fig. 3). Mice of the GEF1, GEF4, and GEF6 lines showed (3 more minor lesions characterized by focal destruction of c fa >,, cardiac myocytes. Gross signs of congestive heart failure, *" M C X ° O such as hepatic and splenic congestion, were observed at m cn I. Ij Ucf -i c. autopsy in some GEF3 animals. The expression of the transgene in all GEF animals was most pronounced in the heart, as measured either by Northern blot analysis of RNA (Fig. 2) or by P1305ag-fPS tyrosine .,.. kinase activity immunoprecipitable from extracts of various l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..-.. ^ ~ ~ ~ _ 28S,' _ tissues (data not shown). To determine whether the severity O ~~~~~~~~~..: I' w i:t .. of cardiac enlargement and injury was related to the levels of P130m4PS, we compared P130ag-PPs autokinase activity im18S munoprecipitable from the hearts of GEF3 and GEF4 animals with heart weight. A direct correlation was observed between the degree of cardiomegaly and the amount of v-fps PTK activity detected in heart lysates of GEF mice (Fig. 4A). Extremely low amounts of P130gaPS were detected in the FIG. 2. Expression of v-fps RNA in a GEF4 mouse. RNA was hearts of mice from the GF2, GF3, and GF6 lines that do not prepared from the indicated tissues of a single 4-month-old GEF4 manifest cardiovascular pathology. mouse or from a normal CD1 mouse and 20 ,ug of each RNA was A formal test of the involvement of the GEF transgene in hybridized on a Northern blot with a v-fps probe. RNA (5 j.g) from cardiomegaly was made by breeding animals of the GEF6 a v-fps-transformed Rat2 cell line (lane Cl-10) served as a positive lineage, in which this phenotype is rather subtle, to homozycontrol. Positions of 18S and 28S rRNAs are shown. gosity. GEF6 homozygotes had a more pronounced cardiocroaching upon the lumen of the left ventricle. At no stage vascular phenotype than their hemizygous siblings, as meawas a polymorphonuclear leukocyte exudate obvious. sured by heart weight (Fig. 4B), and could be instantly A comparison of hearts from animals of GF and GEF lines, distinguished by an increased reddening of the skin. As from normal CD1 mice, or from transgenic mice with an expected, the P130gagfPs tyrosine kinase activity immunounrelated oncogene (p53) was performed blind. The cardiac precipitable from heart lysates of homozygous GEF6 mice pathology was specific for animals with the GEF transgene, was twice that of their hemizygous littermates (Fig. 4B). GEF4

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FIG. 3. Cardiac and trigeminal nerve pathology in GEF transgenic mice. (A) Myocardium of a GEF3 mouse showing the degenerate appearance of myocytes, fibrous replacement, and mononuclear infiltration. (x400.) (B) Myocardium of a normal CD1 mouse. (x400.) (C) Coronal section through the base of the skull of a GEF4 mouse. Arrowheads point to the trigeminal nerves on either side of the sphenoid bone. (x 19.) (D) Corresponding coronal section of a normal CD1 mouse. Arrowheads point to the trigeminal nerves. (x 19.)

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FIG. 4. P130gagfPs kinase activity in hearts of GF and GEF transgenic mice correlates with cardiomegaly. (A) Hearts from GEF3, GEF4, GF2, GF3, or GF6 transgenic animals or from a normal CD1 mouse were dissected, rinsed with PBS, wiped dry, and weighed (heart weight in mg is indicated). Hearts were then homogenized in RIPA buffer, normalized for total protein, and immunoprecipitated with anti-pl9m monoclonal antibody (+) or control monoclonal antibody (-). Autophosphorylation of immunoprecipitated P13091-PS on tyrosine was analyzed in in vitro kinase reactions, followed by SDS/polyacrylamide gel electrophoresis and autoradiography. P13DOPPS immunoprecipitated from a Fujinami sarcoma virus-transformed Rat2 cell line (lane Cl-10) was included as a positive control. The mobilities of phosphorylated P130D8PS and of stained size markers are indicated. K, kDa. (B) Two hemizygous GEF6 animals were mated and their offspring were analyzed for the GEF transgene by Southern analysis and by mating to normal CD1 animals. Hearts from three 4-month-old littermates that were either homozygous (lanes 1 and 2) or hemizygous (lanes 3 and 4) for the GEF transgene or were devoid of transgenic sequences (CD1; lanes 5 and 6) were weighed (mg for individual hearts are indicated). Hearts were then homogenized, normalized for protein, and immunoprecipitated with anti-p19P9 antibody (+) or control antibody (-). P130PPS autophosphorylation was assayed in vitro. Cl-10 cells (lanes 7 and 8) provided a positive control.

Neurological Phenotypes in v-fps Transgenic Mice. All animals of the GF2 line exhibited a marked trembling and hyperactivity. Tremors were not observed in GF3 or GF6 mice but were present in all animals of the GEFi, GEF3, GEF4, and GEF6 lines (see Fig. 5A). v-fps RNA was detected in the brains of animals with tremors (GEF lines and GF2) but was not identified in the brains of GF3 and GF6 animals that lack the trembling phenotype (Fig. 5B). P130PV4PS tyrosine kinase activity was also detected in the brains of trembling mice (data not shown). These data are consistent with a neurological phenotype arising from v-fps expression in the central nervous system. In the peripheral nervous system of mice in the GEF3 and GEF4 lineages, we have observed a dramatic bilateral enlargement of the intracranial segments of the trigeminal nerves (Fig. 3). DISCUSSION We have obtained a series of transgenic mice that express the P130gag4Ps cytoplasmic PTK and display a variety of physiological and pathological abnormalities involving the cardiovascular and nervous systems. By analysis of a number of individual lines, we conclude that these traits are all specific to the transgene. Animals of all the GF and GEF lines discussed above also developed tumors of lymphoid or mesenchymal origin (S.P.Y., D.M., V.M., J.R., A.B., and T.P., unpublished data), consistent with the widespread expression of the transgenic oncogene in lymphoid and other tissues. In contrast to traits such as cardiomegaly and trembling that developed rapidly and showed complete penetrance, tumors developed with a latent period of several months and arose with various frequencies. Apparently additional genetic or epigenetic events are required for tumor formation in GF and GEF mice, whereas cardiac and neurological phenotypes are solely dependent on expression of the v-fps PTK. This difference may reflect cellular restraints on malignant proliferation. The range of phenotypes induced by the GF and GEF transgenes reflects their widespread expression. There is

considerable precedent for anomalous transcription of chimeric transgenes, particularly in the heart and central nervous system (24-27). The patterns of transgene expression are not due to position effects, since they are identical in several different lines. In agreement with previous observations (22), our results indicate that the 5' f3-globin promoter sequence does not confer tissue-specific expression on a nonerythroid gene. An element flanking the human p-globin locus that specifies position-independent 8-globin expression in transgenic mice has been identified (28, 29). This element, which is absent from our constructs, may be required in addition to 5' and 3' f-globin regulatory sequences to confer appropriate erythroid expression on heterologous genes. Cardiovascular abnormalities observed in GEF mice were correlated with v-fps expression in the heart. Myocardial damage in these mice was characterized by atrophy and necrosis of myofibers and their replacement by fibrous connective tissue and was accompanied by the formation of mural thrombi. These myocardial changes induced congestive heart failure or a cardiomyopathy. Although cardiomyopathy in man can have a number of quite distinct causes, the underlying molecular basis for heart damage is not known. The histology of GEF mice did not suggest an infection. The cardiac cell necrosis did not resemble "contraction band necrosis" induced, for example, by catecholamines nor, in the absence of a polymorphonuclear leukocyte exudate, was it truly equivalent to the coagulation necrosis diagnostic of myocardial infarction (30). Nonetheless, the phenotype ofGEF transgenic mice demonstrates that a defined genetic lesion can induce a cardiomyopathy and provides a starting point from which to investigate the molecular mechanisms by which myocardial damage can be inflicted. In contrast to GEF animals, transgenic mice expressing SV40 large tumor antigen in the heart display an unusual myocyte hyperplasia (24, 31). Several observations suggest that myocardial disease in GEF mice is a primary response to expression of the P130gag4Ps in the heart. First, GEF mice expressed high levels of P1309ga-fPs tyrosine kinase activity in the heart relative to

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mice provides direct evidence that PTK expression can affect nervous system function and suggests that PTKs are important in mammalian neural development.

B

GF

GEF

34

We are indebted to G. Jackowski, U. Danilczyk, K. Maruyama, F. Keeley, and R. Renlund for their generous help and for stimulating discussions and to T. Townes for providing the ,3-globin plasmid. We thank K. Hewlett and T. Lee for excellent technical assistance and S. Mackey and M. Postar for preparation of the manuscript. This work was supported by a Terry Fox Programme Project Grant from the National Cancer Institute of Canada. S.-P.Y. is a postdoctoral fellow, and T.P. and J.R. are Terry Fox Cancer Research Scientists of the National Cancer Institute of Canada.

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FIG. 5. Tremors correlate with v-fps expression in the brain. (A) A 4-month-old GEF4 transgenic mouse (on the left) and a normal littermate (on the right) were photographed under stroboscopic illumination (6 flashes per sec) with a 1-sec exposure. (B) Total RNA was isolated from the brains of GEF3, GEF4, GEF6, GF2, GF3, GF6, and CD1 mice. Brain RNA (20 tug) from each animal was analyzed by Northern blot analysis using a v-fps nick-translated probe as described in the legend to Fig. 2. Total RNA (5 ,ug) from Fujinami sarcoma virus-transformed Cl-10 Rat2 cells served as a positive control.

other tissues. In addition, the level of cardiac P130saf4Ps kinase activity in different lines correlated with the extent of cardiomegaly and the severity of myocardial damage. Furthermore, the blood pressures of GEF, GF, and normal CD1 mice were indistinguishable, as measured by cannulation of the aorta at 3 months and 6 months of age, indicating that the cardiomegaly is unlikely to represent a secondary response to pressure overload (S.-P.Y., F. Keeley, and K. Maruyama, unpublished results). The cardiomegaly and accompanying myocardial damage in GEF mice might result from direct synthesis of P130-fPs in cardiac myocytes or from expression of P130-fPs in the nonmyocyte cells. Immunocytochemical analysis of GEF3 hearts with an anti-gag monoclonal antibody revealed specific staining in the mononuclear interstitial cells (unpublished results). Although we cannot rule out the possibility that P130gag-fPs is expressed to low levels in myocardiocytes, these results suggest that the cardiac damage results primarily from expression of P130aI8-Ps in the interstitial cells. These cells proliferate to form fibrotic lesions in GEF hearts and may release paracrine factors that influence myocardiocyte function. v-fps expression in the brain is correlated with the marked trembling exhibited by GF2 and GEF animals. PTKs are particularly abundant in the central nervous system (32) where they are postulated to regulate processes such as myelination (33), responses to neurotransmitters (34), or neurite extension (35). The specific enlargement of the trigeminal nerve in some GEF lines suggests that this peripheral nerve is particularly sensitive to aberrant expression of a tyrosine kinase and may normally respond during development to signals involving tyrosine phosphorylation. The identification of neurological abnormalities in GF2 and GEF

1. Jaenisch, R. (1988) Science 240, 1468-1474. 2. Stewart, T. A., Pattengale, D. K. & Leder, P. (1984) Cell 38, 627-637. 3. Quaife, C. J., Pinkert, C. A., Ornitz, D. F., Palmiter, R. D. & Brinster, R. L. (1987) Cell 48, 1023-1034. 4. Sinn, E., Muller, W., Pattengale, P., Tepler, I., Wallace, R. & Leder, P. (1987) Cell 49, 465-475. 5. Bautsch, V. L., Toda, S., Hassell, J. A. & Hanahan, D. (1987) Cell 51, 529-538. 6. Ruther, U., Garber, C., Komitowski, D., Muller, R. & Wagner, E. F. (1987) Nature (London) 325, 412-416. 7. Williams, R. L., Courtneidge, S. A. & Wagner, E. F. (1988) Cell 52, 121-131. 8. Hunter, T. & Cooper, J. A. (1985) Annu. Rev. Biochem. 54, 897-930. 9. Yarden, Y. & Ullrich, A. (1988) Annu. Rev. Biochem. 57, 443-478. 10. Marth, J. D., Lewis, D. B., Wilson, C. B., Geam, M. E., Krebs, E. G. & Perlmutter, R. M. (1987) EMBO J. 6, 2727-2734. 11. Quintrell, N., Lebo, R., Varmus, H. E., Bishop, J. M., Pettenati, M. J., Le Beau, M. M., Diaz, M. 0. & Rowley, J. D. (1987) Mol. Cell. Biol. 7, 2267-2275. 12. Martinez, P,., Mathey-Prevot, B., Bernards, A. & Baltimore, D. (1987) Science 237, 411-415. 13. Pawson, T., Guyden, J., Kung, T.-H., Radke, K., Gilmore, T. & Martin, G. S. (1980) Cell 22, 767-775. 14. Shibuya, M. & Hanafusa, H. (1982) Cell 30, 787-795. 15. Sadowski, I., Stone, J. C. & Pawson, T. (1986) Mol. Cell. Biol. 6, 43964408. 16. Hogan, B., Costantini, F. & Lacy, E. (1986) Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 17. Auffray, C. & Rougeon, F. (1980) Eur. J. Biochem. 107, 303-314. 18. Maniatis, J., Fritsch, E. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 19. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) J. Mol. Biol. 113, 237-251. 20. Weinmaster, G., Zotler, M. J., Smith, M., Hinze, E. & Pawson, T. (1984) Cell 37, 559-568. 21. Behringer, R. R., Hammer, R. E., Brinster, R. L., Palmiter, R. D. & Townes, T. (1987) Proc. Natl. Acad. Sci. USA 84, 7056-7060. 22. Trudel, M., Magram, J., Bruckner, L. & Costantini, F. (1987) Mol. Cell. Biol. 7, 4024-4029. 23. Trudel, M. & Costantini, F. (1987) Genes Dev. 1, 954-961. 24. Behringer, R. R., Peschon, J. J., Messing, A., Gartside, C. L., Hanschka, S. D., Palmiter, R. D. & Brinster, R. L. (1988) Proc. Natl. Acad. Sci. USA 85, 2648-2652. 25. Alpert, S., Hanahan, D. & Teitelman, G. (1988) Cell 53, 295-308. 26. Russo, A. F., Crenshaw, E. B., III, Lira, S. A., Simmons, D. M., Swanson, L. W. & Rosenfeld, M. G. (1988) Neuron 1, 311-320. 27. Soriano, P., Cone, R. D., Mulligan, R. C. & Jaenisch, R. (1986) Science 234, 1409-1413. 28. Grosveld, F., van Assedelft, G. B., Greaves, D. R. & Kollias, G. (1987) Cell 51, 975-985. 29. Ryan, T. M., Behringer, R. R., Martin, N. C., Townes, T. M., Palmiter, R. D. & Brinster, R. L. (1989) Genes Dev. 3, 314-323. 30. Baroldi, G. (1975) Am. Heart J. 89, 742-752. 31. Fields, L. J. (1988) Science 239, 1029-1033. 32. Letwin, K., Yee, S.-P. & Pawson, T. (1988) Oncogene 3, 621-627. 33. Edwards, A., Arquint, M., Braun, P. E., Roder, J. C., Dunn, R. J., Pawson, T. & Bell, J. C. (1988) Mol. Cell. Biol. 8, 2655-2658. 34. Hopfield, J. F., Tank, D. W., Greengard, P. & Huganir, R. L. (1988) Nature (London) 336, 677-680. 35. Manness, P., Aubry, M., Shores, C. G., Frame, L. & Pfenninger, K. H. (1988) Proc. Natl. Acad. Sci. USA 85, 5001-5005.