Tk+/- mouse model for detecting in vivo mutation in an endogenous, autosomal gene.
Tk+/- transgenic mice were created using an embryonic stem cell line in which one allele of the endogenous thymidine kinase (Tk) gene was inactivated by targeted homologous recombination. Breeding Tk+/- parents produced viable Tk-/- knockout (KO) mice. Splenic lymphocytes from KO mice were used in reconstruction experiments for determining the conditions necessary for recovering Tk somatic cell mutants from Tk+/- mice. The cloning efficiency of KO lymphocytes was not affected by the toxic thymidine analogues 5-bromo-2'-deoxyuridine (BrdUrd) or trifluorothymidine (TFT), or by BrdUrd in the presence of lymphocytes from Tk+/- animals; however, it was easier to identify clones resistant to BrdUrd than to TFT when Tk+/- cells were present. Tk+/- mice were treated with vehicle or 100 mg/kg of N-ethyl-N-nitrosourea (ENU), and after 4 months, the frequency of Tk mutant lymphocytes was measured by resistance to BrdUrd. The frequency of Tk mutants was 22+/-5.9x10-6 in control animals and 80+/-31x10-6 in treated mice. In comparison, the frequency of Hprt mutant lymphocytes, as measured by resistance to 6-thioguanine, was 2.0+/-1.2x10-6 in control animals and 84+/-28x10-6 in the ENU-treated mice. Analysis of BrdUrd-resistant lymphocyte clones derived from the ENU-treated animals revealed point mutations in the non-targeted Tk allele. These results indicate that the selection of BrdUrd-resistant lymphocytes from Tk+/- mice may be used for assessing in vivo mutation in an endogenous, autosomal gene.
Dobrovolsky
V N
VN
Division of Genetic and Reproductive Toxicology, HFT-120, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA. vdobrovolsky@nctr.fda.gov
Casciano
D A
DA
Heflich
R H
RH
D000818:Animals; D003433:Crosses, Genetic; D005038:Ethylnitrosourea / Q000633:toxicity; D005260:Female; D005796:Genes / Q000187:drug effects*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008957:Models, Genetic; D016296:Mutagenesis; D013937:Thymidine Kinase / Q000172:deficiency* / Q000235:genetics*
D007041:Hypoxanthine Phosphoribosyltransferase; D013937:Thymidine Kinase; D005038:Ethylnitrosourea