Parameters governing the transfer of the genes for thymidine kinase and dihydrofolate reductase into mouse cells using metaphase chromosomes or DNA

Abstract

The conditions necessary to achieve high frequency transfer of the thymidine kinase and dihydrofolate reductase genes from hamster cells into mouse cells were investigated. Of the parameters examined, the length of adsorption time, input gene dosage, and treatment with dimethylsulfoxide (DMSO) were found to significantly alter the transfer frequency using either metaphase chromosomes or purified DNA as the transfer vehicle. With the mouse cell line as a recipient, the optimal adsorption period for DNA or chromosomes from MtxRIII cells was found to vary from 8 to 16 h in those experiments where the recipient cells were subsequently treated with DMSO. Without DMSO, similar frequencies could be obtained by extending the period of adsorption. Increasing the dosage of DNA or chromosomes resulted in an almost linear increase in the number of transformants. The optimal conditions for transfer did not significantly differ for the two genes studied. On the average, the optimal conditions yielded 1.5 x 10(3) transformants per 10(7) recipient cells with chromosomes; with DNA an average of only 60 transformants were observed. In general, DNA transformants grown in the absence of methotrexate were unstable; whereas, under the same conditions about 20% of the transformants from the chromosome experiments were stable.

En bloc incorporation of coatomer subunits during the assembly of COP- coated vesicles [published erratum appears in J Cell Biol 1994 Jul;126(2):589]

“…Transfection of DNA into CHO cells was performed by the calcium phosphate coprecipitation method as described (Lewis et al, 1980). One ml of calcium phospbate/DNA precipitate containing 22/~g of pSVSport.…”

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity.

“…Metaphase chromosomes were purified from hydroxyurea-resistant cell lines as previously described (31). Chromosomes were precipitated with calcium phosphate (12) and added to recipient cells by a modified procedure (14,16 Two-dimensional electrophoresis of cell extracts. Cells exponentially growing in 150-cm2 tissue culture flasks were washed once with ice-cold sonication buffer consisting of 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 mM Tris-hydrochloride (pH 6.8).…”

“…4 and Table 2). (11,16,27). A total of eight HydR transformant colonies were picked from the selection plates, grown to 107 cells in the presence of hydroxyurea, and then passaged in drug-free culture medium for a period of 100 cell doublings (ca.…”

Transfer of Murine Leukaemia and Murine Sarcoma Virus Genetic Information by Transfection with Isolated Metaphase Chromosomes

“…A similar observation was made by Butch & McBride (1975) for small variations in the input ratio of chromosomes to recipient cells. However, Lewis et al (1980) have noted some effect of chromosome dosage. To determine whether the foci produced after chromosome treatment represent true Ha-MuSV transformants and not the spontaneous foci which sometimes arise in normal cells (Shih et al, 1979), the treated cultures were tested for their ability to form transmissible Ha-MuSV(MuLV) pseudotype particles.…”

“…The use of isolated metaphase chromosomes to transfer genetic information into cultured mammalian cells has been demonstrated by a number of different laboratories (Burch & McBride, 1975;Cassingena et al, 1978;Lewis et al, 1980;McBride & Ozer, 1973;Miller & Ruddle, 1978;Mukherjee et aL, 1978;Shani et al, 1974;Shih et al, 1979;Willecke & Ruddle, 1975). Although the genome of the DNA tumour virus, SV40, has been transferred by this technique (Cassingena et al, 1978;Shani et al, 1974), successful transfer of retrovirus genomes with this procedure has not been reported to date.…”