Proc. Nati. Acad. Sci. USA Vol. 82, pp. 6927-6931, October 1985 Cell Biology Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection (preimplantation mouse embryos/germ-line integration/DNA methylatlon/5-azacytidine/gene expression) DETLEV JAHNER*t, KIRSTEN HAASE*, RICHARD MULLIGANt, AND RUDOLF JAENISCH*t *Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie an der Universitat Hamburg, Martinistr. 52, 2000 Hamburg 20, Federal Republic of Germany; and tWhitehead Institute for Biomedical Research, and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142 Communicated by Robert A. Weinberg, July 1, 1985 ABSTRACT Mouse substrains genetically transmitting the Retroviral vectors have been constructed that can exogenous Moloney murine leukemia virus (Mo-MuLV) at a transduce foreign genes into a variety ofcell types (12-22). In single locus have been derived previously by infection of previous experiments, a vector transducing a bacterial mark- preimplantation embryos. Here we explore the potential of er gene has been introduced into postimplantation mouse retroviral vectors for transferring nonviral genes into the germ embryos by injection of virus, and the transduced gene was line ofmice. Preimplantation mouse embryos were cocultivated shown to be active in many somatic tissues ofanimals derived with a cell line that produces a recombinant retrovirus whose from injected embryos (17). Viral insertion into germ line genome carries the Escherichia cofi gene gpt. We show that the cells was, however, not obtained in these experiments. vector sequence was inserted into the genome of the embryo To explore the potential of retroviral vectors to transfer and into the germ line at a frequency similar to that for the nonretroviral genes into the germ line of mice, we have Mo-MuLV-helper sequence. A new mouse strain, Mgpt-1, was exposed preimplantation embryos to MSVgpt virus, which is developed that is homozygous for a single MSVgpt proviral a Mo-MuLV-derived vector carrying the Escherichia coli genome. The proviral sequences were highly methylated and gene gpt. We show that the vector is inserted into germ-line not expressed in tissues of Mgpt-1 mice. When cells derived cells at a similar frequency as Mo-MuLV helper virus. The from transgeneic animals were treated with 5-azacytidine, the inserted gene is highly methylated and transcriptionally proviral sequences were not methylated and were transcrip- inactive but can be activated by treatment of cells with tionally activated. These results indicate that nonviral genes 5-azacytidine. that are under the control of the viral long terminal repeat are inactivated when transferred into the germ line of animals. MATERIALS AND METHODS The transfer of foreign genes into an animal's germ line represents a powerful tool for the study of gene regulation Mice. Embryos were derived from mice of strain 129. The during mammalian development. To date, the primary meth- origin of Mov-9 and Mov-13 mice has been described (8). od employed to generate transgeneic mice has involved the Cell Lines and Infection of Embryos. NIH 3T3, XC, and microinjection of recombinant DNA into the zygote Cl-1-lA cells were grown as described (23). The gpt-7-3T3 pronucleus (1-5). Although resulting mouse strains show cell line, derived by infection of NIH 3T3 cells with MSVgpt variable expression of the inserted gene, in a number of virus and Mo-MuLV, has been described (24). Four-to-eight- instances the expected pattern of tissue-specific expression cell mouse embryos were isolated and infected by cocultiva- was observed. A potential alternative to the method of tion with gpt-7-3T3 cells as described (7, 8). Primary Cell Culture. Lung tissue of Mgpt-1 mice was microinjection involves the exposure of early embryos to sliced, rinsed in Dulbecco's phosphate-buffered saline, and retroviruses (6-8). In contrast to the complex, tandemly incubated for 60 min at 370C in Dulbecco's modified Eagle's repeated structure that is frequently observed after introduc- medium supplemented with 10% fetal bovine serum and with tion of DNA into the germ line by microinjection, retroviral and cells infection of preimplantation embryos leads to the stable 0.25% collagenase (Sigma). Bone marrow spleen well-defined that can be were prepared as described (25). integration of single, genomes gpt Virus Assay. Primary lung fibroblasts (2 x 10W) were analyzed easily at the DNA and RNA levels as well as by infected with Mo-MuLV from Cl-i-lA cells. Two weeks assays for viral infectivity. Exposure of preimplantation later, 2 x 10i of these cells were cocultivated for 3 days with mouse embryos to Moloney leukemia virus (Mo-MuLV) 5 x results in efficient integration of proviral sequences into the 105 NIH 3T3 cells in the presence of Polybrene (Aldrich) derived and were subsequently tested for growth in selective medium embryo's genome and into the germ line of animals containing mycophenolic acid (25 ,ug/ml) as selective drug from the infected embryos (6-8). The viral sequences are de (26). novo methylated soon after infection and are not expressed Similarly, 107 bone marrow or spleen cells were coculti- (9). Upon further development of the embryo, these inserted with 2 in vated for 3 days in the presence of Polybrene x 10 proviral sequences remain methylated and suppressed NIH 3T3 cells in RPMI 1640 medium/10% fetal bovine serum most differentiated cells (9). In contrast, retroviruses intro- supplemented with either 10% supernatant from WEHI-3 duced into postimplantation embryos can replicate in an cells (for bone marrow cells) or 50 gg of bacterial lipopoly- unrestricted manner and remain unmethylated in all somatic These media were tissues ofthe developing embryo (for review, see refs. 10 and saccharide per ml (for spleen cells). kindly 11). Abbreviations: Mo-MuLV, Moloney murine leukemia virus; MSVgpt virus, a Mo-MuLV-derived vector containing the Esche- The publication costs of this article were defrayed in part by page charge richia coli gene gpt; SV40, simian virus 40; wt, wild-type. payment. This article must therefore be hereby marked "advertisement" tPresent address: Salk Institute, P.O. Box 85800, San Deigo, CA in accordance with 18 U.S.C. §1734 solely to indicate this fact. 92138. 6927 Downloaded by guest on September 28, 2021 'orf7%28Cell Biology: Rihner et al. Proc. Natl. Acad. Sci. USA 82 (1985) by T. Franz (Heinrich-Pette-Institut). Three days B provided A E P+ S X Pv con- later, the cells were tested for growth under selective a b c d ditions (26). -E } Analysis of DNA and RNA. All restriction enzymes were obtained from Boehringer Mannheim or from Bethesda Research Laboratories and used as recommended by the supplier. Preparation of high molecular weight DNA, elec- trophoresis in 0.8% agarose gels, blotting to nylon mem- branes, and hybridizations were as described (27). Plasmid pSV2 (24) was used to detect gpt and simian virus 40 (SV40) sequences. A BamHII-HindIII fragment from plasmid pL10 (24) served as a gpt-specific probe. al (I) collagen sequences were detected with plasmid HF677 (28). The synthesis of a "Dot cDNA specific for Mo-MuLV has been described (7). .~~~~~~SS i blot" hybridizations were performed with 5 jxg of DNA prepared from tail, and RNA was prepared and analyzed as described (28). gpt [Xanthine (Guanine) Phosphoribosyltransferase, EC 2.4.2.22] Assay. Extracts were prepared from 2-10 x 106 cells by repeated freezing and thawing in 50 mM Tris Cl (pH 7.5). Extract (10 jig of protein) was incubated for 60 min at 370C (29), using ['Cixanthine (Amersham) instead of [14C]gua- FIG. 1. (Upper) Restriction enzyme analyses of mouse 100-19 nine. The amount of [14C]xanthosine 5'-phosphate was de- and its offspring. pSV2 (24) was the hybridization probe (see termined by binding to DEAE plates and measurement of Materials and Methods). (A) EcoRI- and Sst I-digested DNAs from radioactivity in a liquid scintillation counter. liver ofmouse 100-19 (lanes a and b, respectively) and from tail ofone of his first-generation offspring (lanes c and d, respectively). EcoRI does not cleave the MSVgpt provirus (see band E); Sst I cuts in the RESULTS long terminal repeats (open bars in map) to yield bandS (see fragment S above map). Due to incomplete transfer of larger fragments to the nylon membrane, bands generated by EcoRI were less intense than Germ-Line Integration of an MSVgpt Proviral Genome and those derived by Sst I digestion of the same amount of DNA. (B) Derivation of Mice Homozygous at the Mgpt-1 Locus. Analysis of the MSVgpt provirus (see map) in DNA from 100-19 Preimplantation mouse embryos were cocultivated overnight offspring (lanes a) and from the gpt-7-3T3 cell line (lanes b) by the with gpt-7-3T3 cells (7, 8). This cell line produces both enzymes indicated above each pair of lanes: E, EcoRV; P, Pst I; 5, MSVgpt and Mo-MuLV helper virus, the latter at a 10-fold Sst I; X, Xba I; Nv, Pvu II. (Lower) Restriction map of MSVgpt excess (see Table 4). Seven mice were derived from infected provirus. Restriction sites are abbreviated as described above; and tail DNAs were analyzed for the presence of additional sites: A, Ava I. The gpt sequence within the provirus (24) embryos the hatched bar. proviral sequences. Three mice were shown to carry only is indicated by while one animal had Mo- Mo-MuLV helper sequences, named MuLV helper as well as MSVgpt proviral sequences inte- sion of the MSVgpt genome. This proviral locus was grated in its genome. This animal (no. 100-19) was bred with Mgpt-1. normal mice to test for genetic transmission of proviral To derive a mouse strain homozygous for the Mgpt-1 sequences.