© 2005 American Heart Association, Inc.
Letters to the Editor |
Troubles With a Transgene: Experiences With SM22
-tTA Mice
Division of Cell & Molecular Biology (T.A., C.W., M.H.), Toronto General Hospital Research Institute, Departments of Medicine (M.A.M., A.-M.S.),, Physiology, and Laboratory Medicine & Pathobiology (S.H., M.H.), Heart & Stroke Richard Lewar Centre for Excellence in Cardiovascular Research, University of Toronto, Canada
In response:
Dr David Dichek and colleagues recount their disappointing experience with doxycycline (Dox)-responsive SM22
-tTA "driver" mice provided to them. In constructing these animals, a 2.8-kb fragment of the SM22 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-ßgal), generously provided by L. Hennighausen.3 In 3 of 12 lines generated, nuclear X-gal staining was seen only in SM22-tTA+/tetO7-ßgal+ mice, was responsive to Dox, and was consistently restricted to tissues known to support expression of the SM22 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 SM22-tTA to drive expression of a dominant negative c-Myb (MEn) and ß-gal. As previously detailed, we screened 7 MEn:ß-gal lines to discover 2 that could be activated by SM22-tTA.5 MEn expression, ß-gal activity, and the phenotypes documented were tTA-dependent, Dox-responsive, identical in both lines, and consistent with results of previous non–tissue-specific strategies targeting c-Myb.5 We next screened 8 lines harboring a transgene encoding human PMCA4b and ß-gal to find 2 that could be activated by SM22-tTA.6 Again, hPMCA4b expression and the phenotype found was tTA-dependent, Dox-responsive, consistent in both lines,6 and near identical to that reported independently.7 No less rigor and precision were applied to the analysis of expression and phenotype of another line 36 SM22-tTA-dependent project published elsewhere.8
Together, the above studies clearly indicate that a functional tTA was expressed in mice provided to Dr Dichek and colleagues. We can think of only a few possible explanations for why they were unable to observe tTA-dependent transgene expression with the same line, at the same time: (1) In their effector and reporter lines, the levels of SM22-driven tTA were insufficient to drive detectable expression of their transgenes. The need to screen several potential effector lines to find those that can be activated by tTA is well known5,6,9; (2) Their dose and duration of Dox therapy were greater than what we later came to use. In both the MEn and hPMCA4b projects, Dox was used primarily in control groups treated with Dox. Once absence of embryonic lethality was confirmed, we did not routinely use Dox-suppression and withdrawal; (3) The animals studied were older than those in which we typically characterize phenotypes. In 2004, we observed in SM22-tTA+/MEn:ßgal+ mice in the LDLr–/– background, that there was no detectable expression of MEn or ß-gal after 3 to 6 months of a high-fat diet. Possible explanations for this included age-, diet/disease-, or strain-dependent repression of SM22 promoter activity.
In 2004, we submitted a manuscript to Circulation Research accounting the phenotype of line 36 SM22-tTA–dependent iNOS overexpression. Earlier, these data and our experience with a project involving line 36 SM22-tTA–dependent ET-1 overexpression were also presented at a Gordon Conference.10 In both these projects, the levels of SM22-tTA–driven transgene expression were much lower than those observed with the corresponding lines in projects using MHC-tTA mice,11,12 generously provided by G. Fishman.13 As a corollary, levels of expression of the hPMCA4b:ß-gal transgene are much higher in crosses with MHC-tTA14 than those reported with SM22-tTA.6 In both venues, we carefully detailed how the iNOS and ET-1 phenotypes were transient in nature with many elements being lost after 8 to 12 weeks of age or postnatal transgene activation. Together, these projects further suggested age-, effector-, or disease-dependent suppression of SM22-tTA.
Many investigators have studied regulatory elements of the SM22 and other SMC-specific promoters,1,15–19 but certain points deserve mentioning. First, as elegantly demonstrated by Regan et al, there exist dramatic differences between SM-MHC promoter-driven ß-gal expression versus the "integrated" signal observed with SM-MHC promoter-driven Cre-mediated activation of a constitutive ß-gal locus (ROSA26-ß-gal).18 Thus, the heterogeneity of expression of transgenes driven by the SM22 promoter may mirror in part the spatially and temporally stochastic nature of VSMC-specific gene expression. Second, key elements within the SM22 promoter mediate loss of expression of genes under its regulation in disease states such as atherosclerosis.19 Indeed, the remarkable phenotypic plasticity of SMC per se is known to be accompanied by changes in the levels of expression of its so called "marker" genes.20 Accordingly, when we observed heterogeneous expression of SM22-tTA–driven transgenes under certain conditions and in specific crosses, we believed these instances primarily to be a function of these variables. However, in retrospect, they may also have been early signs of a more systematic "silencing."
In trying to respond to our Reviewers’ comments on the iNOS project, we have found that line 36 SM22-tTA can no longer activate the iNOS transgene. Recent analyses show that our SM22-tTA lines no longer exhibit detectable levels of tTA expression by RT-PCR. Why independent SM22-tTA integration sites, previously active, have gone silent has regrettably become an area of our research. We do not believe this represents strain-dependent loss, inadvertent breeding-out, or rearrangement of a previously active integration site, as the SM22-tTA transgene has been in the C57Bl6 background for years, and we have not discerned changes by Southern blot. Our current hypothesis is that these lines have experienced progressive silencing of either the SM22 promoter or the bacterial and viral sequences encoding tTA, as a consequence of epigenetic factors that we hope to elucidate and report. Indeed, while many investigators anecdotally comment on the failure of various transgenes over time, a systematic accounting or analysis of these events and the mechanisms underlying them is lacking. Moreover, given our experiences with SM22-tTA mice, and other data regarding the SM-MHC-Cre animal,21,22 we wonder whether SMC-specific gene expression may be particularly prone to this phenomenon. As we rederive SM22-tTA animals, we are constructing newer reagents designed to be insulated from potential epigenetic modifications. Fortunately, other VSMC-specific conditional gene expression systems have been generated,23,24 which should, in a timelier manner, meet the needs of the cardiovascular research community for these valuable tools.
Acknowledgments
Dr Husain is a Career Investigator of the Heart & Stroke Foundation of Ontario (HSFO). This work was supported in part by operating grants from the Canadian Institutes of Health Research (MOP-14648 & 117801) and the HSFO (T5254).
References
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