SV40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA

• Published in: Gene Vol. 66; no. 1; pp. 65 - 76
• Main Authors: Mayne, Lynne V., Jones, Tina, Dean, Stephen W., Hal-court, Susan A., Lowe, Jill E., Priestley, Anne, Steingrimsdottir, Herdis, Sykes, Hazel, Green, Michael H.L., Lehmann, Alan R.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 01.01.1988; Amsterdam Elsevier; New York, NY
• Subjects: Ataxia-telangiectasia; Biological and medical sciences; Biotechnology; Cockayne's syndrome; DNA repair; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; genomic DNA; Methods. Procedures. Technologies; recombinant DNA; transfection; Vectors (cloning, transfer, expression). Insertion sequences and transposons; xeroderma pigmentosum; Cockayne's syndrome; genomic DNA; UV; SDS; transfection; ori; A-T; xeroderma pigmentosum; CHO; DNA repair; bp; CS; R; Ataxia-telangiectasia; MEM; G418; u; Hyg; kb; SV40; XP; recombinant DNA; SSC 0.15 M NaCl; Human; Cockayne syndrome; Transformation; Transformed cell; Deficiency; Polyomavirus; Molecular integration; Xeroderma pigmentosum; Papovaviridae; Genetic transfer; Virus; Resistance; Antibiotic; Cell line; Repair; SV40 virus; Fibroblast
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(88)90225-9

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0–40 kb in length. The transfection frequency for the selected (G418 R) marker was between 2 × 10 −4 and 2 × 10 −3 for all cell lines, comparable to many other mammalian systems. About 50% of the G418 R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.