The application of the restriction site mutation assay to compare 1-ethyl-1-nitrosourea-induced mutations between the endogenous p53 gene and the transgenic LacZ gene in MutaMouse testes

Transgenic mouse modelling has provided a new approach to study the various steps involved in spontaneous and induced mutagenesis in rodent somatic ac id germline tissues in vivo. However, the important question arises as to w hether mutations occur at the same rate in transgenes as in endogenous gene s. Here, the restriction site mutation (RSM) assay was used to study mutati ons induced in the endogenous p53 gene and LacZ transgene of MutaMouse test es treated with 1-ethyl-1-nitrosourea (ENU), The aim of these experiments w as to compare mutation susceptibility between the endogenous p53 gene and t he integrated LacZ gene in the transgenic mouse. ENU-treated and control te stes were analysed 102 days after treatment; a total of 297 RSM analyses we re performed on ENU-treated and untreated testis DNA, Ten mutational events were detected in the p53 gene (exon 5 and intron 8), two of which occurred in untreated animals and probably represent spontaneous events. Only a sin gle mutation was detected in the LacZ gene of an ENU-treated animal by the RSM assay. Thus the RSM assay can readily detect ENU-induced mutations in t he p53 gene, but not in the LacZ transgene. Comparison of the LacZ RSM muta tion data with results from a previous study of identically dosed MutaMice in the transgenic selection assay [Ashby,J., Gorelick,N.J. and Shelby,M.D. (1997) Mutat. Res., 388, 111-122] showed that LacZ mutations were far more readily recovered with the MutaMouse transgenic selection assay than by RSM analysis, The reason for the relative inability of the RSM assay to detect LacZ mutations may be the smaller target size of the RSM analysis compared with the transgenic selection assay (16 bases compared with 3000 bases). T aking into account the different target sizes by calculating the mutation f requency per base allowed the RSM data regarding p53 and LacZ to be compare d with previously published data from transgenic selection assays. These st udies demonstrated that the p53 mutations were present at mutation frequenc ies (per base) 5- to 70-fold higher than the LacZ gene mutations, In additi on, the LacZ mutation frequency per base found in the RSM was an order of m agnitude higher than that found in the transgenic selection assay. The tran sgenic selection assay is more sensitive per locus (due to the larger targe t of the LacZ gene), as evidenced by ability to detect ENU-induced testes m utations readily.