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

Abstract

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 transgenic mice. 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 beta-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/tTA(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 beta-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.

Deviation of islet autoreactivity to cryptic epitopes protects NOD mice from diabetes

“…Transgenic mice carrying tTA under control of the ICA69 or the CMV promoter have been generated in our lab, but in both lines tTA‐transgene expression was progressively silenced. Similar observations have recently been reported in other tTA‐transgenics where some inherent tTA toxicity was noted 34, 35. Our data demonstrate that it was possible to generate persistent tolerance to the major Tep69 epitope in NOD mice.…”

A previously functional tetracycline-regulated transactivator fails to target gene expression to the bone

“…However, speculative reasons for defective tetracycline-regulated activators have been discussed before [20,21]. Long-term instability because of epigenetic modification, such as methylation [27,28], problems related to small construct sizes, positional- and dosage-dependent effects [25], and unexpected transgene expression pattern [29] have been reported by various investigators [3,22]. The tet-ON/OFF system is a powerful model system for studying genes in vitro or in vivo .…”

Unexpected Expression Pattern of Tetracycline-Regulated Transgenes in Mice

“…Failure of tet-based strategies in vivo has also been reported. This has been related either to defective tTA expression (Bö ger and Gruss 1999; Fedorov et al 2001;Lee et al 2006) or to epigenetic repression on the tet-promoter activity ( Janicki et al 2004;Pankiewicz et al 2005;Kues et al 2006), in addition to undesired interactions of eukaryotic cell factors with the tetpromoter sequences (Rang and Will 2000;Gould and Chernajovsky 2004).…”