Sa. King et al., XERODERMA-PIGMENTOSUM VARIANT - GENERATION AND CHARACTERIZATION OF FIBROBLASTIC CELL-LINES TRANSFORMED WITH SV40 LARGE T-ANTIGEN, Experimental cell research, 217(1), 1995, pp. 100-108
Xeroderma pigmentosum variant (XPV) fibroblasts from the XP4BE strain
(CRL1162) were transformed with the SV40 large T antigen with the purp
ose of generating immortalized cell lines that are defective in postre
plication repair (PRR). Two transformation and selection protocols wer
e used and at least two independent clones were obtained, which behave
d in culture as immortal cell lines, Fingerprinting analyses were used
to demonstrate their origin from XP4BE cells and to compare their gen
etic profiles. These cell lines were shown to be hypersensitive to kil
ling by uv when compared to SV40-transformed fibroblasts derived from
foreskins of normal neonates, One of the XPV transformed cell lines (C
Tag) was characterized further as a potential source of cell-free extr
acts with capability for catalyzing the T antigen-dependent in vitro r
eplication of plasmid DNA carrying the SV40 origin of replication. In
this assay system, CTag extracts were shown to be as active as those p
roduced from HeLa cells. In vitro replication of uv-damaged plasmid DN
A by protein extracts from PRR-defective (XPV) and PRR-proficient cell
s might allow the identification and characterization of protein facto
rs that contribute to normal replication of uv-damaged DNA. Ultraviole
t irradiation of plasmid DNA templates caused dose-dependent inhibitio
n of in vitro replication by both HeLa and CTag extracts. (C) 1995 Aca
demic Press, Inc.