Transfection Of Genomic DNA Research Articles

Induction of cell surface insulin antigenicity in fibroblasts transfected with islet genomic DNA

Ltk −cells were transfected with total genomic DNA obtained from rat islets or from insulinoma cells mixed with DNA tagged with fluorescent dye. Flyorescence-activated cell sorter (FACS) was used for initial selection of successful transfectants, monitoring fluorescent DNA incorporated into the cell. For subsequent selections the cells were treated with anti-insulin antiserum labeled with fluorescent dye and selected by FACS. B cell DNA, tiut not DNA from murine leukemic cells, induced the appearance of cell surface insulin antigenicity in fibroblasts. This phenotypic expression was transient. Together with our previous demonstration of the induction of insulin secreation in Ltk − cells [(1986) Biochem. Biophys. Res. Commun. 136, 638-644], these results indicate that B cell specific characteristics can be transfected to non-endocrine cells by genomic DNA transfection.

Double-strand break repair and G 2 block in Chinese hamster ovary cells and their radiosensitive mutants

Two X-ray-sensitive mutants of the CHO K1 cell line were examined for their cell-cycle progression after irradiation with γ-rays, and for their ability to rejoin double-strand breaks (DSBs) as detected by neutral filter elution. Both mutants were impaired in DSB rejoining and both were irreversibly blocked in the G 2 phase of the cell cycle as determined by cytofluorometry. From one mutant we have isolated several revertants. The revertants stem from genomic DNA transfection experiments and may have been caused by gene uptake. All revertants survived γ-irradiation as did the wild-type CHO line. One of them has been examined for its ability to rejoin DSBs and was found to be similar to the wild type.

Cooperation between Multiple Oncogenes in Rodent Embryo Fibroblasts: an Experimental Model of Tumor Progression?

Publisher Summary Stepwise progression of tumoral diseases toward increased invasiveness, hormonal independence, and resistance to chemical and physical treatment is an all-too-common phenomenon that has been analyzed in various experimental systems. The stepwise character and apparent irreversibility of these phenotypic changes would be most easily accounted by a sequential accumulation of genetic alterations. Alterations of cellular genes associated with malignancy (oncogenes) are recently identified, first as a result of the natural capacity of retroviruses for gene transduction and subsequently by the transfection of genomic DNA from human tumors into mouse cells. Unlike most retroviruses, oncogenic DNA viruses that have been analyzed so far appear to require more than one gene to establish and maintain neoplastic transformation. This was extensively documented for polyoma viruses and adenoviruses, and more recent observations suggest a similar situation for two herpesviruses— herpes simplex type 2 and Epstein–Barr virus. Experimental evidence summarized in this chapter defines an early stage of the oncogenic transformation process induced by the plt and E1A oncogenes and, at least under some defined conditions of expression, by the rearranged forms of the myc gene.

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

New Acceptor Cell for Transfected Genomic DNA: Oncogene Transfer into a Mouse Mammary Epithelial Cell Line

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

Activation of nonexpressed endogenous ecotropic murine leukemia virus by transfection of genomic DNA into embryo cells.

We studied the infectivity of endogenous ecotropic murine leukemia virus genomes contained in high-molecular-weight DNA prepared from virus-free cells of the AKR-2B line, and from RF, BALB/c, B6, and (BALB/c x B6)F(1) mouse embryo cells. When DNA prepared from virus-free AKR-2B cells was transfected into NIH-3T3 cells, no virus-positive cultures were observed, a result consistent with previous reports. However, when DNAs from virus-free AKR-2B cells or virus-free cells containing the RF/J or BALB/c ecotropic proviruses were transfected into chicken embryo cells that were then cocultivated with SC-1 (mouse) cells, virus-positive cultures were recovered. The specific infectivities of the AKR provirus(es) contained in virus-free cells and the molecularly cloned Akv-1 provirus were similar when chicken embryo cells were used as primary recipients. Virus-positive cultures were also observed when secondary mouse embryo cells were used as recipients for DNA from virus-free AKR-2B and RF/J cells. The transfected chicken embryo-SC-1 cultures produced XC-positive murine leukemia virus that is N-tropic. Virus-positive recipient cultures were observed 10- to 100-fold more frequently when AKR-2B DNA was used than when BALB/c DNA was used as the donor DNA. Our studies indicate that some nonexpressed ecotropic murine leukemia virus proviruses are activated upon transfection into chicken embryo cells. Such studies suggest that there are different factors governing the expression of murine leukemia virus after transfection into established cell lines (NIH-3T3) and into nonestablished secondary cultures (chicken and mouse).