Generation dependent reduction of tTA expression in double transgenic NZL-2/tTA^CMV mice

Despite the overall successful application of the tet-system to regulate gene expression in vitro and in vivo, nothing is known so far about the long-term stability of this system in transgenicmice. In this study, mice of generation F2, F3, F4, or F10 of two independent tTA^CMV transgenic lines were bred with NZL-2 mice containing a tTA-responsive bidirectional promoter that allows the simultaneous expression of two reporter genes encoding luciferase and β-galactosidase. Analysis of the expression of transgenes in double transgenic mice revealed a dramatic reduction of tTA transactivator mRNA over time. As a consequence, the expression of both reporter genes was gradually reduced from generation to generation in tissues of embryonic and adult NZL-2/t TA^CMV mice. Luciferase activity in NZL-2/ tTA^CMV(F10) mice was reduced 8-10-fold compared to NZL-2/ tTA^CMV(F2) mice, and β-galactosidase expression was no longer detectable. In summary, we describe the long-term instability of the tet-system in our NZL-2/tTA^CMV double transgenic mice. The molecular basis of this observation and experimental tools to overcome this limitation need to be addressed in future.