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 +/- 5.9 X 10(-6) in control animals and 80 +/- 31 X 10(-6) in treated mice. In comparison, the frequency of Hprt mutant lymphocytes, as measured by resistance to 6-thioguanine, was 2.0 +/- 1.2 X 10(-6) in control animals and 84 +/- 28 X 10(-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. (C) 1999 Elsevier Science B.V. All rights reserved.