Quantification of Expression of Linked Cloned Genes in a Simian Virus 40-Transformed Xeroderma Pigmentosum Cell Line

Abstract

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).

Use of a Simian Virus 40-Based Shuttle Vector to Analyze Enhanced Mutagenesis in Mitomycin C-Treated Monkey Cells

“…To determine whether excision repair of UV-damaged templates might play a role in enhanced mutagenesis, we determined the capacity of excision repair-deficient xeroderma pigmentosum group A (XPA) cells (XP12BeSV40; obtained from the Human Genetic Mutant Cell Repository, Camden, N.J. and grown in Dulbecco minimal essential medium with antibiotics and 10% fetal bovine serum) to exhibit enhanced mutagenesis after pretreatment with mitomycin C. In this case, the amount of mitomycin C used to treat the cells and the UV fluence used to treat the vector had to be reduced to compensate for the increased sensitivity of XPA cells to these agents. The calcium phosphate-DNA precipitation method was used for transfection as described previously (19). The results in Table 2 indicate that enhanced mutagenesis occurred in XPA cells, and therefore this phenomenon probably does not involve excision repair.…”