Transgene stability and features of rasH2 mice as an animal model for short-term carcinogenicity testing

The transgenic mouse rasH2 line, in which the mouse carries the human c-Ha-ras gene under the control of its own enhancer and promoter, has been proposed as one of the alternative short-term models for carcinogenicity testing. To apply this purpose, we have produced a genetically homogeneous population as C57BL/6JJic-TgN(RASH2) (Tg-rasH2) by continuous backcrossing. In this study, we examined the transgene stability between different generations and the detailed transgene architecture of the integrated human c-Ha-ras gene. Fluorescence in situ hybridization analysis showed that the integrated human c-Ha-ras gene was stably located on chromosome 15E3 in Tg-rasH2 mice at generation number (N) 15 and 20. Southern and Northern blot analysis did not show any differences in the hybridized band pattern in each generation. Southern blot analyses showed that the Tg-rasH2 mouse contained three copies of the human c-Ha-ras gene arrayed in a head-to-tail configuration. We also determined the nucleotide sequence of the transgene in the Tg-rasH2 mouse at N20 and confirmed that the sequence of the coding region was perfectly matched with human c-Ha-ras cDNA. Cloning and sequencing of genome/transgene junctions revealed that integration of the microinjected human c-Ha-ras gene into mouse host genome resulted in a 1820-bp deletion in the rasH2 line. The deleted sequence did not have any sequence homologies with known functional genes. We assumed that either the deletion or the transgene insertion, or both, would not cause insertional mutation. In short-term carcinogenicity testing with a genetically engineered mouse model, confirmation of the transgene or modified gene stability at each generation is one of the important factors that affect the sensitivity to carcinogenic compounds in the same way as the genetic background, age and route of administration.