Failure to detect a DNA repair-related defect in the transfection of ataxia-telangiectasia cells by enzymatically restricted plasmid.
We have transfected two SV40-transformed human fibroblast cell lines with plasmids in which double-strand breaks have been introduced by restriction enzymes, within or near the selected gene. Restriction of pSV2gpt with KpnI, as previously shown by Cox et al. (1986), reduced the frequency of transfection more in the ionizing radiation-sensitive ataxia-telangiectasia line AT5BIVA than in the resistant line MRC5V1. When the related plasmid pSV2neo was restricted with SmaI, the reduction in transfection was less in the ataxia-telangiectasia than in the normal cell line. Under our conditions, the apparent defect in transfection of AT5BIVA by pSV2gpt appears to be a result of the unusual sensitivity of the repair-deficient recipient to the selective agent. Loss of potential transfectants is exacerbated when transient gene expression is reduced by restriction of the plasmid. We suggest that a reduction in yield of transfectants with restricted plasmid in ataxia-telangiectasia cells cannot readily be used as evidence of a defect in DNA repair. Our results are also relevant to standard transfection experiments, since they emphasize the importance of optimizing selection when transient expression may be reduced, to ensure that potential transfectants are not killed by the selection regime.
D001260:Ataxia Telangiectasia; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D010957:Plasmids / Q000187:drug effects*; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D004262:DNA Restriction Enzymes