DNA-mediated gene transfer without carrier DNA

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.     

DNA-mediated transfer of a human DNA repair gene that controls sister chromatid exchange.

The Chinese hamster cell line mutant EM9, which has a reduced ability to repair DNA strand breaks, is noted for its highly elevated frequency of sister chromatid exchange, a property shared with cells from individuals with Bloom's syndrome. The defect in EM9 cells was corrected by fusion hybridization with normal human fibroblasts and by transfection with DNA from hybrid cells. The transformants showed normalization of sister chromatid exchange frequency but incomplete correction of the repair defect in terms of chromosomal aberrations produced by 5-bromo-2'-deoxyuridine.     

Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells.

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.     

DNA-mediated transfer and expression of a human DNA repair gene that demethylates O6-methylguanine.

Human liver DNA was transfected into CHO cells (mex-) along with pSV2gpt and colonies were selected first for resistance to mycophenolic acid and then to chloroethylnitrosourea. Transformants were obtained that contained approximately 10,000 molecules of O6-alkylguanine alkyltransferase (mex+) per cell. Their genome contained at least three copies of the human Alu sequence.     

DNA-mediated gene transfer into epidermal cells using electroporation

A reliable method for the introduction of foreign DNA into epidermal cells is described. Electroporation of murine BALB/c MK-1 epidermal cells with pSV2-CAT resulted in the transient expression of chloramphenicol-acetyltransferase (0.03 to 0.05 nmoles acetylchloramphenicol per mg protein per min) in the transfected cells. Transfection of MK-1 cells with pSV2-neo led to the appearance of approximately eight G418 resistant clones per 10(-6) cells per microgram of plasmid DNA. Distinct patterns of integration of SV2-neo were detected in three different resistant clones.     

Radiation-induced DNA damage and its repair

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes. A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.     

DNA damage by peplomycin and its repair in an in vitro system.

1. DNA damage by peplomycin, an antitumor antibiotic, and its repair by cellular enzymes were studied using pUC18 plasmid DNA. The DNA damage and repair were measured by monitoring the conformational changes of pUC18 DNA. 2. Peplomycin-induced DNA damage was enhanced by addition of ferrous ion and inhibited by deferoxamine, a specific iron chelator, suggesting iron-requirement for the DNA damage. 3. DNA damage by peplomycin was inhibited by superoxide dismutase in both native and heat-inactivated forms, possibly due to non-enzymatic interaction. 4. Peplomycin-induced, single-strand breaks in pUC18 DNA was repaired by incubating with a priming factor (an exonuclease purified from mouse ascites sarcoma cells), DNA polymerase beta, four deoxynucleoside triphosphates, T4 DNA ligase and ATP. The average repair patch size was estimated to be approximately four nucleotide length.     

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.     

Verapamil enhances the efficiency of DNA-mediated gene transfer in mammalian cells

Treatment of Ltk^? cells with the calcium antagonists, verapamil and diltiazem, but not nifedipin, causes a 3-fold enhancement of the frequency of transfer of the cloned gene for herpes simplex virus thymidine kinase (HSV-tk). The frequency of phenotypic expression of the HSV-tk DNA was 20 to 34 times higher than that of genotypic transformation. Phenotypic expression was also 2.3 to 2.6 times increased when 20 μg/ml of verapamil was present during calcium phosphate-mediated DNA transfection.     

Unusual aspects of human thymidylate synthase in mouse cells introduced by DNA-mediated gene transfer

Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme. In two transformants examined, however, neither was the dTTP pool elevated nor the dCTP pool decreased. DNA synthesis in permeabilized cells of a transformant was more efficient than that in the wild type with dATP, dGTP, dCTP, and dUMP as substrates, but this was not so when dUMP was replaced by dTTP. Unlike the mouse enzyme, the human enzyme in the transformants did not co-sediment with DNA polymerase alpha and thymidine kinase in a sucrose gradient, suggesting that the human enzyme is not incorporated into a multienzyme complex for DNA replication. The high levels of the human enzyme in the transformants were suppressed to various degrees by fusion with a wild type mouse line. No active hybrid dimer enzyme was found between the human and mouse enzymes, which each consist of two identical subunits. Thus, the human enzyme in the transformants seems to behave differently from the mouse enzyme and its overproduction seems to be necessary for supporting the normal growth of the transformants.     

Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function

A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk^?) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 × 10⁶ D) was used as a carrier, the presence of either 20 mM NH₄Cl, 1 μM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-P_i) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 × 10⁶ D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH₄Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-P_i coprecipitate, whereas NH₄Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.     

DNA-mediated gene transfer in Chinese hamster ovary cells: Clonal variation in transfer efficiency

Summary Thymidine kinase-deficient Chinese hamster ovary (CHO) cells were genetically transformed with the BamHI restriction fragment encoding the thymidine kinase gene of herpes simplex virus (HSV-tk). We have observed considerable clonal variation among independent CHO sublines with respect to transformation competence for the DNA-mediated gene transfer of HSV-tk. Transformation frequencies 3×10^-4 were observed consistently in one subline, with a transformation efficiency of approximately 1 transformant per ng viral gene. The frequency and efficiency of transformation we observed in this system are at least 10-fold greater than those previously reported for DNA-mediated transformation of CHO cells by HSV-tk. All of the CHO HSV-tk⁺ transformants examined were stable for the transferred genotype in the absence of selection, and all showed evidence of co-transformation by unselected plasmid pBR322 sequences.A preliminary account of these results was given at the ICN-UCLA Symposia, March 21–28, 1982     

Pesticide induced DNA damage and its repair in cultured human cells.

The effects of pesticides on the induction of unscheduled DNA synthesis in SV-40 transformed human cells (VA-4) in culture with and without metabolic activation by liver microsomes was studied. Results showed that ten of the thirteen compounds examined either directly or upon metabolic activation induced unscheduled DNA synthesis in the human cell system used. The DNA repair kinetics and size of the repaired regions resulting from treatment with four of the chemicals (Carbaryl, Chlordane, Dieldrin and 2.4-D Fluid) were studied by 313 nm photolysis of repaired regions containing bromodeoxyuridine (BUdR). The size of the repaired regions differed between compounds but could generally be classified as either of the X-ray (short) or UV-type (long).     

Bleomycin-induced DNA damage and DNA repair in chicken embryo cells as compared to X-irradiation

Following in vitro- and in ovo-exposure of chicken embryo cells, the level of bleomycin (BM)-induced damage was evaluated by using DNA synthesis, nucleoid sedimentation (SED), and viscometry of alkaline cell lysates (VISC). This damage was compared to X-irradiation, using 5.9-378 nM BM in vitro, 1.5-116 micrograms BM/egg in ovo, and 2-32 Gy, respectively, in vitro as well as in ovo. With respect to BM, the most notable result is the increase in DNA synthesis and VISC at the lowest concentrations of the drug. A decrease in both parameters was observed at high BM concentrations and following exposure to X-rays, concomitantly with an increase in SED. Regarding the radiomimetic drug BM and X-rays, different modes of DNA damage and DNA repair are suggested by previous investigations and the present results. Therefore, further evidence is presented, that the chicken embryo can act as a simple, rapid and inexpensive test system to characterize the biological effects of many nucleo- and/or cytotoxic agents.     

Use of electroporation for high-molecular-weight DNA-mediated gene transfer

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.     

The effect of X-rays and ultraviolet light on DNA-mediated gene transfer in mammalian cells

The uptake, expression and genomic integration of exogenous DNA during DNA-mediated gene transfer are poorly understood in mammalian cells. We studied the effects of ionizing radiation and u.v. light treatments on recipient cells during gene transfer experiments. We found that both X-rays and u.v. light stimulate pSV2-gpt DNA transfer into V79 Chinese hamster cells and they are equally effective for an equi-cytotoxic dose. This result was observed with irradiation both before and after the period of DNA precipitate overlay of the recipient cells. The stimulation of DNA transfer was approximately proportional to dose for both types of radiation. The effect was significantly enhanced using chronic, rather than acute, radiation treatments. The optimal expression time to observe stimulation of DNA transfer, however, differs for the two radiation types. A possible model for DNA-mediated gene transfer, incorporating this result, is discussed.     

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer.

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.     

DNA-mediated transfer of an RNA polymerase II gene: reversion of the temperature-sensitive hamster cell cycle mutant TsAF8 by mammalian DNA

Treatment of the TsAF8 temperature-sensitive (TS) mutant of Syrian hamster BHK-21 cells, with calcium phosphate precipitates of genomic TS+ DNAs from a variety of mammalian cell lines permitted the selection of TS+ colonies at 40 degrees C. TS+ transformation events were distinguished from spontaneous TS+ reversions in experiments in which alpha-amanitin-sensitive (Amas) TS+ DNA was used to transform an AmaR derivative of TsAF8 cells and AmaR TS+ DNA was used to transform Amas TsAF8 cells. In each case it was possible to demonstrate the unselected acquisition of the appropriate Amas or AmaR phenotype with the selected TS+ allele. Each of these TS+ transformed cell lines when grown at 40 degrees C contained an RNA polymerase II activity with a sensitivity to inhibition by alpha-amanitin characteristic of the particular DNA used to transform the TS cells, whereas at 34 degrees C the same cells contained a mixture of AmaR and Amas polymerase II activities. Together, these data provide convincing evidence that the RNA polymerase II gene determining sensitivity to inhibition by alpha-amanitin can be transferred to TsAF8 cells and that the TS defect in TsAF8 is a polymerase II mutation.     

DNA-mediated transfer of cAMP resistance in CHO cells

Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP-dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild-type CHO cells by DNA-mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8-Br-cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP-dependent protein kinase was fractionated on a DEAE-cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild-type cells, also failed to phosphorylate a 52,000-dalton protein in a cAMP-dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP-dependent protein kinase has been transferred. The ability to transfer this gene by DNA-mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP-dependent protein kinase.