A new retroviral vector for detecting mutations and chromosomal instability in mammalian cells.
A retroviral vector carrying both forward (neo) and backward (herpes simplex virus thymidine kinase or HSV-TK gene) selection markers was constructed as a substrate for mutational assay in mammalian cells. The cells infected with this virus are first selected with G418, mutagenized and then selected with the anti-herpes drug acyclovir (ACV). Since HSV-TK, but not the host TK, is capable of converting ACV to a toxic metabolite, cells retaining the intact HSV-TK gene fail to survive, while the cells carrying a mutated HSV-TK gene or which have lost the gene can form colonies in the presence of ACV, making it possible to detect the genetic defects in a positive manner. It is also possible to discriminate between small mutations and large deletions by checking the presence of the linked marker, neo. As a model experiment, we prepared an uncloned pool of rat fibroblast cells (CREF) infected with this virus and irradiated them with increasing doses of ultraviolet light. Dose-dependent increases in the number of ACV-resistant colonies were observed. Structural analysis of the HSV-TK gene in these clones revealed point mutations or small deletions in the majority of the cases. Since it requires no pre-existing genetic markers in the host cells, this system may be used for a wide variety of mammalian cells and provides a useful tool to assess both their susceptibility to various mutagens and their genomic instability.
Murata
S
S
Department of Dermatology, Jichi Medical School, Tochigi, Japan.
Matsuzaki
T
T
Takai
S
S
Yaoita
H
H
Noda
M
M
D000212:Acyclovir / Q000378:metabolism; D001355:Alpharetrovirus / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009152:Mutagenicity Tests / Q000379:methods*; D051381:Rats; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014466:Ultraviolet Rays / Q000009:adverse effects
D005839:Gentamicins; C010680:antibiotic G 418; D013937:Thymidine Kinase; D000212:Acyclovir