Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line.
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).
Protić-Sabljić
M
M
Whyte
D
D
Fagan
J
J
Howard
B H
BH
Gorman
C M
CM
Padmanabhan
R
R
Kraemer
K H
KH
D000123:Acetyltransferases / Q000235:genetics; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002460:Cell Line; D002472:Cell Transformation, Viral; D015500:Chloramphenicol O-Acetyltransferase; D003001:Cloning, Molecular; D004260:DNA Repair; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D010430:Pentosyltransferases / Q000235:genetics; D011995:Recombination, Genetic; D013539:Simian virus 40; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase