A recombinant plasmid composed of segments of herpes simplex virus and simian virus 40 viral DNA inserted into the bacterial plasmid pBR322 was microinjected into pronuclei of fertilized mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by the Southern blotting technique for the presence of DNA homologous to the injected plasmid. Two of 78 mice in one series of injections showed clear homology, though the injected sequences had been rearranged. Band intensities from the two positive mice were consistent with the presence of donor DNA in most or all of the cells of the newborns. These results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos. This technique affords the opportunity to study problems of gene regulation and cell differentiation in a mammalian system by application of recombinant DNA technology.

Rejection of bone marrow grafts in irradiated mice is mediated by natural killer (NK) cells and is controlled by genes linked to the major histocompatibility complex (MHC). It has, however, not been possible to identify the genes or their products. An MHC class I (Dd) transgene introduced in C57BL donors prevented the rejection of their bone marrow by NK cells in irradiated allogeneic and F1 hybrid mice expressing the Dd gene. Conversely, H-2Dd transgenic C57BL recipients acquired the ability to reject bone marrow from C57BL donors but not from H-2Dd transgenic C57BL donors. These results provide formal evidence that NK cells are part of a system capable of rejecting cells because they lack normal genes of the host type, in contrast to T cells, which recognize cells that contain abnormal or novel sequences of non-host type.

Coprecipitation of DNA with calcium phosphate is a commonly used method of gene transfer in mammalian cells. We have found that DNA forms a tight complex with Ca P. and that DNA in this complex is resistant to nucleases present in serum or added externally. Under optimal conditions, virtually all of the recipient mouse LtklAprt-cells take up Ca P.-DNA complexes, as determined by fluorescent dyes specific for DNA (4',6-diaminilo-2-phenylindol dihydrochloride) or for calcium salts (chlorotetracycline). However, only a small proportion of the cells have detectable Ca Pi-DNA complexes in the nucleus. Uptake of the Ca P-DNA complexes was highly dependent upon the pH at which the Ca P.-DNA complex was formed and upon the concentration of DNA in the complex.It has been known for many years that under certain conditions, eukaryotic cells can take up large amounts ofDNA and transport it into the cell nucleus (1, 2). This phenomenon has been exploited to achieve transformation of eukaryotic cells with both selectable and nonselectable genes (1, 2, ¶). Addition of polycations, such as polyornithine (3) or DEAE-dextran (3, 4), to the DNA solution or precipitation of the added DNA on the cell surface by calcium phosphate (5) have proven useful in increasing both the uptake ofthe DNA and the frequency ofcell transformation. Despite the large amount of high molecular weight DNA taken into cells in the presence of certain facilitators (3, 6), at-best only about one in 104 cells eventually becomes transformed (7,8). This has limited transfer to those genes for which there exists a good selective system and to certain cell lines, such as mouse L cells, that function as efficient recipients (8).The detailed mechanisms by which external DNA is integrated into a foreign chromosome and expressed in a foreign environment are as yet poorly understood. However, a large body of recent work (9, 10) has begun to elucidate the process. On the other hand, essentially nothing-is known about the way by which DNA molecules cross the cellular membrane and are transported to the nucleus. The studies described here were designed to study the quantitative aspects and the mechanisms of entry of DNA into cells after coprecipitation with Ca Pi. We have used the specific fluorescent dye 4',6-diaminilo-2-phenylindol dihydrochloride, (DAPI), which specifically stains double-stranded DNA (11), and chlorotetracycline, which stains complex salts ofCa (12). We show that virtually all recipient cells take up Ca Pf-DNA complexes, which appear in the cytoplasm in a characteristic structure, and that only a few ofthe cells have detectable Ca P,-DNA complexes in the nucleus. Additionally, DNA uptake was highly dependent on the pH at which the Ca Pi precipitate was formed and upon the concentration of DNA in that, precipitate. MATERIALS AND METHODSCell Culture. The murine cell line Ltk-Aprt-was cultured in minimum essential medium, a modification (GIBCO), sup-plemented with 10% (vol/vol) fetal calf serum. In most cases, cells were plated in 35-mm or 60-mm...

Two plasmids containing nonoverlapping deletions of the herpes simplex virus thymidine kinase gene were introduced into thymidine kinase-deficient mouse L cells by DNA-mediated gene transfer. Thymidine kinase-producing transformants were generated by a mixture of the two plasmids at a frequency significantly greater than that generated by either plasmid alone. Southern blot analyses demonstrated that functional thymidine kinase genes were generated by homologous recombination between the two deletion mutants.

JC virus and BK virus are ubiquitous human viruses that share sequence and structural homology with simian virus 40. To characterize tissue-specific expression of these viruses and to establish model systems for the study of human viral-induced disease, transgenic mice containing early regions of each of the viruses were produced. The viral sequences induced tumors in a distinct and tissue-specific manner that was similar to their tissue tropism in humans. Ten JC virus-containing founder mice were produced, of which 5 survived to maturity. Four of them developed adrenal neuroblastomas, which metastasized to several other tissues. JC virus tumor-antigen RNA was detected at high levels in the tumor tissues and at low levels in the normal tissues of these mice. One of the three BK virus-containing mice was abnormally shaped and died at 2 weeks of age. The other two BK virus-containing mice developed primary hepatocellular carcinomas and renal tumors and died at 8-10 months of age. BK virus tumor-antigen RNA was expressed in tumor tissues of both mice. Since each of the viruses retained the general tissue tropism that it exhibits in humans, these data suggest that transgenic mice harboring human viruses will be useful as animal models for viral-induced diseases.

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The H-2Dd transgenic strain D8 on C57BL background was more resistant to subcutaneous challenge of RBL-5 lymphoma cells than B6 controls. The direct role of the H-2Dd antigen was investigated by the use of (D8 x B6)F1 crosses and (D8 B6) x B6 backcrosses. The latter showed cosegregation with regard to Dd antigen expression and lymphoma resistance, both of which were inherited in a pattern consistent with control by a single dominant gene. The rejection potential in (D8 x B6)F1 mice appeared as strong as that seen in crosses between B6 and MHC congenic mice (on B10 background) carrying H-2Dd. The lymphoma resistance could be abrogated by treatment with anti-asialo GM1 antiserum or anti-NK 1.1 mAb, indicating a role for NK cells.