Troubles With a Transgene: Experiences With SM22α-tTA Mice

Abstract

In response: Dr David Dichek and colleagues recount their disappointing experience with doxycycline (Dox)-responsive SM22α-tTA “driver” mice provided to them. In constructing these animals, a 2.8-kb fragment of the SM22α promoter, generously provided by J. Miano and E. Olson,1 was placed upstream of a tetracycline-responsive (Tet-OFF) transcriptional activator (tTA), generously provided by M. Gossen and H. Bujard.2 Screening of founders was conducted in crosses with G3 reporter mice (tetO7-βgal), generously provided by L. Hennighausen.3 In 3 of 12 lines generated, nuclear X-gal staining was seen only in SM22α-tTA+/tetO7-βgal+ mice, was responsive to Dox, and was consistently restricted to tissues known to support expression of the SM22α promoter in development, young adults (typically assessed <12 weeks of age), and after arterial injury. Although X-gal staining was patchy, varying in intensity between lines (#21, 19, 36, from weakest to strongest) and at different points in the arterial tree, it mirrored the fold-amplification of tTA-dependent Luciferase activity in tetO7- luc reporter mice and correlated with levels of tTA expression, as determined by RT-PCR.4 Regarding articles appearing in Circulation Research , we first used line 36 SM22α-tTA to drive expression of a dominant negative c-Myb (MEn) and β-gal. As previously detailed, we screened 7 MEn:β-gal lines to discover 2 that could be activated by SM22α-tTA.5 MEn expression, β-gal …