Regulated Transgene Expression in Vascular Smooth Muscle
To the Editor:
We are writing in reference to 2 articles published in Circulation Research, both of which report use of transgenic mice with smooth muscle cell–specific expression of the tetracycline-regulated transactivator (tTA) to activate conditional tTA-regulated alleles in the vasculature of transgenic mice.1,2 The purpose of this letter is to report our experience with these mice and to inform the readership that, in our hands, these mice show no evidence of either (1) an ability to transactivate conditional alleles or (2) expression of the tTA in vascular tissue.
Generation of mice in which a fragment of the murine SM22α promoter was used to achieve smooth muscle–specific expression of the tTA was first reported in abstract form in 1997 by Husain et al.3 In 1998, experimental results obtained with these mice appeared in a figure contributed by Dr Husain to a review article in Circulation Research.4 This figure depicted doubly transgenic SM22α-tTA/tetO-β-galactosidase (β-gal) embryos and aortas harvested from adult doubly transgenic SM22α-tTA/tetO-β-gal mice. Incubation of the embryos with X-gal chromogen showed doxycycline-suppressible β-gal expression primarily in the heart and somites, with apparent faint staining in the vasculature. The X-gal–stained aortas showed sparse β-gal expression which was “inhibited” by doxycycline treatment.
Based on these reports, we requested these transgenic SM22α-tTA mice for use in our own investigations. In 1999 Dr. Husain kindly sent us transgenic SM22α-tTA mice derived from 2 independent lines. We were particularly appreciative of this generosity because at this time the SM22α-tTA mice had not yet been reported in a peer-reviewed original research publication. To our surprise, however, we could not detect a functional SM22α-tTA allele in these mice. Therefore, in 2000 we visited Dr Husain’s laboratory, reviewed the data that his laboratory had obtained with the SM22α-tTA mice, and presented our negative results to his group. Based on the data that we viewed at that time, we continued our work with the aforementioned 2 lines. We were also provided with a third, independently-generated line of SM22α-tTA mice, which were said to have higher expression of the tTA than the other 2 lines.
In extensive experiments performed largely during the year 2000, but extending into 2001 to 2002, we found no evidence that the SM22α-tTA mice could transactivate either of 2 tTA-regulated alleles that were proven, in our laboratory and elsewhere,5 to be transactivated by the tTA expressed in αMHC-tTA transgenic mice (kindly provided by Dr Glenn Fishman).6 We also found no evidence that the SM22α-tTA mice expressed detectable levels of tTA in the aorta either by Western or Northern analysis. Since this time, 3 articles have appeared in which these SM22α-tTA mice are reported to express tTA (based on RT-PCR, not by Northern or Western analysis) and to transactivate conditional alleles in the vasculature of adult mice.1,2,7 Our uniformly negative results, which do not appear to be attributable to problems with assay sensitivity, contrast with these positive reports. Whether our results cast any doubt on the validity of these reports is best addressed by the authors of these reports.
We are submitting the primary data that report our experience with the SM22α-tTA mice elsewhere, for consideration for publication as a peer-reviewed research article. We are optimistic that these data will be published in this format,* which would allow our fellow scientists to examine our methods and results closely, and compare them with the published record.1,2,7 For now, this letter makes our central conclusions available promptly and directly to readers of the 2 articles in Circulation Research. These conclusions are likely of interest to the large number of investigators who are interested in generating or obtaining mice that have regulated transgene expression in the vasculature. We hope that this letter will stimulate further work which eventually leads to the generation of SM22α-tTA (or similar) mice that are uniformly useful.
This work was supported by grants from the National Institutes of Health to D.A.D. (HL61860 and HL69063). S.L. and R.A. were supported by T32 HL07731 (to UCSF). M.K. and A.D.F. were supported by T32 HL07828 (to the University of Washington). A.D.F. was also supported by a fellowship from the Everest Foundation.
↵*Note added in proof The primary data are in press: Lee S, Agah R, Xiao M, Frutkin AD, Kremen M, Shi H, Dichek DA. In vivo expression of a conditional TGF-β1 transgene: no evidence for TGF-β1 transgene expression in SM22-tTA transgenic mice. J Mol Cell Cardiol. In press.
You XM, Mungrue IN, Kalair W, Afroze T, Ravi B, Sadi AM, Gros R, Husain M. Conditional expression of a dominant-negative c-Myb in vascular smooth muscle cells inhibits arterial remodeling after injury. Circ Res. 2003; 92: 314–321.
Gros R, Afroze T, You XM, Kabir G, Van Wert R, Kalair W, Hoque AE, Mungrue IN, Husain M. Plasma membrane calcium ATPase overexpression in arterial smooth muscle increases vasomotor responsiveness and blood pressure. Circ Res. 2003; 93: 614–621.
Husain M, Olson E, Rosenberg R. Conditional transgene expression in arterial smooth muscle. Circulation. 1997; 96 (suppl I): I-501.
Fishman GI. Timing is everything in life. Conditional transgene expression in the cardiovascular system. Circ Res. 1998; 82: 837–844.
Yu Z, Redfern CS, Fishman GI. Conditional transgene expression in the heart. Circ Res. 1996; 79: 691–697.
Ju H, Gros R, You X, Tsang S, Husain M, Rabinovitch M. Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice. Proc Natl Acad Sci U S A. 2001; 98: 7469–7474.