MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2521-2527 0270-7306/90/062521-07$02.00/0 Copyright C 1990, American Society for Microbiology

Vol. 10, No. 6

Myeloid Expression of the Human c-fps/fes Proto-Oncogene in Transgenic Mice PETER GREER,* VICTOR MALTBY, JANET ROSSANT, ALAN BERNSTEIN, AND TONY PAWSON Division of Molecular and Developmental Biology, Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto, Ontario MSG IX5, Canada Received 21 August 1989/Accepted 13 February 1990

The mammalian c-fpslfes proto-oncogene encodes a 92-kilodalton cytoplasmic protein-tyrosine kinase (p92c`fes), which is expressed in immature and differentiated hematopoietic cells of the myeloid lineage. To determine the limits of the c-fpslfes locus and to investigate the cis-acting sequences required to direct appropriate tissue-specific expression, a 13-kilobase-pair fragment of human genomic DNA containing the entire c-fps/fes coding sequence was introduced into the mouse germ line. Transcription of the human c-fpslfes transgene was highest in bone marrow and showed a tissue distribution identical to that of the endogenous mouse gene. Macrophages cultured from transgenic mouse bone marrow contained particularly high levels of human and murine c-fpslfes RNA. Furthermore, expression of human c-fpslfes RNA induced a proportionate increase in the level of the p92c-fs protein-tyrosine kinase in bone marrow, bone marrow-derived macrophages, and spleen. Elevated levels of normal human p92c-fes had no obvious effect on mouse development or hematopoiesis. Remarkably, given the short 5'- and 3'-flanking sequences, expression of the human proto-oncogene in bone marrow was independent of integration site, was proportional to the transgene copy number, and was of comparable efficiency to that of the endogenous mouse c-fpslfes gene. The 13-kilobase-pair fragment therefore defines a genetic locus sufficient for the appropriate tissue-specific expression of the fps/fes protein-tyrosine kinase and includes a dominant cis-acting element that directs integration-independent myeloid expression in transgenic mice.

Proto-oncogenes have been implicated in the regulation of cell differentiation and may be important in the control of embryonic development (2, 9, 14, 21, 26, 27, 32). In most cases, however, the physiological activities of vertebrate proto-oncogenes and the mechanisms by which their expression is controlled remain untested. Several mammalian proto-oncogenes show a very restricted pattern of expression, suggesting that their transcription is tightly regulated and that their products have lineage-specific functions. The mammalian c-fpslfes gene encodes a 92-kilodalton cytoplasmic protein-tyrosine kinase (p92c-fes) that has only been detected in hematopoietic cells, predominantly in immature myeloid progenitor cells and in differentiated granulocytes and macrophages (4, 18). Low levels of p92c-fes have also been detected in some B-cell lines (18). Similar observations have been made for the corresponding avian gene (25). High levels of c-fpslfes RNA and protein are found in human myeloid leukemias, presumably reflecting the expansion of a cell population in which the c-fpslfes gene is transcriptionally active (4, 11, 18, 28). These observations suggest that the p92c-f'es protein-tyrosine kinase is involved in regulating proliferation and differentiation during myelopoiesis. Although c-fpslfes expression is confined to hematopoietic cells, oncogenic v-fpslfes alleles have been the most frequently isolated retroviral transforming genes. High-level ectopic expression of normal c-fpslfes proteins in fibroblasts does not generally result in transformation (5, 11), suggesting that structural alterations are required for constitutive kinase and transforming activities. Indeed, c-fpslfes proteins can be enzymatically and oncogenically activated by the N-terminal addition of retroviral gag sequences (5, 7). The signals that normally trigger p92cfes activity in vivo have not been discerned. Since p92c-fes, like the src and abl protein*

tyrosine kinases, lacks any transmembrane or extracellular domain, it is not expected to itself bind a hormone but might reversibly associate with other membrane or cytoskeletal components as part of a signal transduction pathway. The introduction of cloned DNA directly into the mouse genome has provided a means to clearly define genetic loci and to explore their functions within the context of the whole organism. To investigate the physiological role of the cfpslfes gene and the elements that regulate its transcription, we used zygote microinjection of genomic human c-fpslfes DNA to generate transgenic mice that display appropriate tissue-specific expression of the human proto-oncogene. MATERIALS AND METHODS Generation of transgenic animals. The 13-kilobase-pair (kb) EcoRI fragment containing the human c-fpslfes gene was purified away from vector sequences (pBR322) by sucrose gradient velocity centrifugation. The DNA was concentrated by ethanol precipitation and dissolved in 1 mM Tris-acetate0.1 mM EDTA (pH 7.5) at a concentration of 2.5 ,ug/ml. Microinjection and oviduct transfer procedures were done by the method of Hogan et al. (16). Embryos for microinjection were derived from CD-1 mice (Charles River Breeding Laboratories, Inc., Wilmington, Mass.). The GEF8 transgenic mouse line used as a control in some experiments expresses the Fujinami sarcoma virus-encoded P13Vag-fps protein under the control of the human 3-globin promoter (35). Identification of transgenic animals. Standard procedures were used for digestion of DNA with restriction endonucleases and agarose gel electrophoresis (19). Transgenic animals were detected by hybridization analysis of DNA isolated from tail biopsy specimens. Samples (2 F.g) were digested with SstI and resolved by agarose gel electrophoresis. After denaturation and renaturation, the agarose gel

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FIG. 1. Human c-fpslfes locus. The 13-kb EcoRI fragment was used to generate transgenic mice by microinjection of CD-1 zygotes. The apparent transcriptional start site (P. Greer, unpublished data) is indicated by a horizontal arrow above the open box designated exon 1. Coding regions of c-fps/fes exons 2 through 19 are represented as filled boxes, with the positions of translational initiation and termination codons indicated. Flanking sequences not included in the transgene are indicated with broken lines. The 3' exon of the closely linkedfur gene

is illustrated to the left of the c-fpslfes locus.

was dried and hybridized with the 32P-labeled human genomic EcoRI c-fpslfes fragment. Final washing was at 65°C in 0.1x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Under these conditions, the endogenous murine locus was not detected. RNase protection analysis. RNA was purified by the acid guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (3). Samples of 5 ,ug of total RNA were subjected to RNase protection analysis (15). 32P-labeled RNase protection probes were synthesized with T7 RNA polymerase from two fragments of the human c-fpslfes gene cloned into pGEM-2 (Promega Biotec, Madison, Wis.). A human-specific probe (PstI probe) was generated from a 90-base-pair (bp) PstI-to-NaeI fragment derived from the noncoding portion of exon 19. A second overlapping probe (AflII probe), which can simultaneously detect and distinguish the human and murine c-fpslfes transcripts, was generated from a 360-bp AfllI-to-NaeI fragment which contains additional upstream sequences including the coding portion of exon 19 and part of intron 18 (see Fig. 3A). Western blot (immunoblot) analysis. Whole tissues were homogenized in RIPA buffer (10 mM Tris hydrochloride [pH 8.0], 100 mM NaCl, 1 mM EDTA, 1% [vol/vol] Nonidet P-40, 0.5% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] sodium dodecyl sulfate) containing protease inhibitors (100 ,ug of phenylmethylsulfonyl fluoride per ml and 50 ,ug of leupeptin per ml). Lysates were clarified by centrifugation at 14,000 x g for 5 min, and aliquots were denatured into sodium dodecyl sulfate sample buffer. Bone marrow cells and cultured cells were washed in Tris-buffered saline and lysed directly in hot sodium dodecyl sulfate sample buffer. Protein concentrations were determined with a Lowry protein assay kit (P-5656; Sigma Chemical Co., St. Louis, Mo.), and 100-,ug samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (17). Western blotting (31) analysis was done with affinity-purified rabbit antiserum raised against a bacterial trpE-c-fpslfes fusion protein containing 45 amino acids of p92c-fes sequence (residues 406 to 451 of human p92c-fs) (24). Antibody-antigen complexes were detected either with 0.2 ,uCi of '25I-protein A (NEX-146L; Dupont, NEN Research Products, Boston, Mass.) per ml or with goat antirabbit-alkaline phosphatase conjugate (Sigma) followed by a color reaction. Immune complex kinase assay. Bone marrow was recovered from femurs and tibias of transgenic or sex-matched nontransgenic litter mates. An equal number of cells (4 x 107) were washed in Tris-buffered saline containing 100 ,uM sodium orthovanadate and lysed with kinase lysis buffer (20 mM Tris hydrochloride [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% [vol/vol] Nonidet P-40, 0.5% [wt/vol] sodium deoxycholate, 100 ,uM sodium orthovanadate, 100 p.g of leupeptin per ml). p92c-fes was immunoprecipitated with crude rabbit antiserum raised against the trpE-c-fpslfes bacterial fusion protein described above. Immune complexes

were then incubated with [-y-32P]ATP and MnCl2 as previously described (11). Cell culture. Rat-2 fibroblasts (30) and human acute myelogenous leukemia (AML) cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. The human AML cell line (OCI-2) was obtained from Mark Minden. The derivation of the human c-fes-expressing rat-2 cell line (EE3/6) has been described previously (11). The murine macrophage cell line BAC1.2F5, obtained from Richard Stanley (20), was grown in alpha modified Eagle medium supplemented with 10% fetal bovine serum, 20% L-cellconditioned medium (as a source of colony-stimulating factor 1), 292 ,ig of glutamine per ml, 20 ,ug of asparagine per ml, and 50 ,uM 2-mercaptoethanol at 37°C in a 5% CO2 atmosphere. For bone marrow-derived macrophage cultures, 5 x 106 bone marrow cells were isolated from femurs and plated on 6-cm plastic tissue culture dishes in 5 ml of BAC1.2F5 medium. Fresh medium was added after 2 days. At 4 days postplating, nonadherent and trypsin-sensitive cells were removed by three 5-min incubations with 0.25% trypsin-EDTA solution at 37°C. The remaining trypsinresistant adherent cells were scraped free, passaged, and grown for an additional 5 days with fresh medium provided every second day. Morphological examination after MayGrunwald-Giemsa staining showed the resulting cells to be an essentially homogeneous population of macrophages. These bone marrow-derived macrophage cells were then used for protein and RNA analysis. RESULTS Generation of transgenic mice carrying the human c-fps/fes gene. The human c-fpslfes gene shown in Fig. 1 has been previously cloned from a human genomic library (12) and completely sequenced, with the exception of approximately 1 kb at the 3' end (22). The proposed intron-exon structure is based on comparison with known avian and feline viral sequences (22) and c-fps/fes cDNAs (33; P. Greer, unpublished data). The gene consists of 18 coding exons (numbered 2 through 19) with a single first noncoding exon. In the human genome, the 3' end of the neighboringfur gene, which shares the same transcriptional orientation as c-fps/fes but is expressed in a distinct set of tissues, is located approximately 1 kb upstream of the presumed c-fps/fes transcription initiation site (22, 23). This raised the possibility that cisacting elements regulating c-fpslfes expression were closely linked to the transcribed sequences. To test this notion, we microinjected the 13-kb EcoRI fragment shown in Fig. 1 into mouse zygotes. Seven transgenic lines were established that stably transmitted the exogenous human c-fps/fes gene. The number of copies of the transgene, determined by DNA hybridization analysis, varied from 4 to 16 (Fig. 2A; Table 1). The transgenes of lines 2 through 7 behaved as autosomal

VOL. 10, 1990

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The mammalian c-fps/fes proto-oncogene encodes a 92-kilodalton cytoplasmic protein-tyrosine kinase (p92c-fes), which is expressed in immature and diff...
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