Targeted Rearrangement of Floxed Alleles in Smooth Muscle Cells in Vivo
To the Editor:
In their biologically and technically important article “Development of a Smooth Muscle-targeted Cre Recombinase Mouse Reveals Novel Insights Regarding Smooth Muscle Myosin Heavy Chain Promoter Regulation”,1 Regan et al reported generation of mice that express cre recombinase from a fragment of the smooth muscle myosin heavy chain (SMMHC) promoter. The biological importance of this article was that it provided strong evidence of either temporally or spatially restricted expression of the SMMHC promoter in smooth muscle cells in vivo. The paper also had two important technical aspects. First, it demonstrated that expression of a SMMHC promoter-cre construct in cre-indicator mice was a more sensitive means of detecting promoter activity than a construct in which the SMMHC promoter expressed lacZ. Second, the SMMHC-cre mice themselves were said to “provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth-muscle-specific gene activation or inactivation.”
Although the SMMHC-cre mice reported by Regan et al have been used by one group for gene activation,2,3 we—as well as other groups that shared their results with us4—found that these mice do not efficiently inactivate floxed alleles in smooth muscle lineages.4 In addition, we found that the cre expression in these SMMHC-cre mice was not restricted to SMC lineages. Specifically, our efforts to use the SMMHC-cre mice to inactivate a floxed allele for the type 2 transforming growth factor beta receptor (tgfbr2flox)5 yielded the following results: (1) SMMHC-cre mice inefficiently rearranged both tgfbr2flox and the cre reporter allele R26R6; (2) The SMMHC-cre transgene was expressed at low levels in smooth muscle-rich organs such as the bladder, stomach, and aorta and at high levels in the testis; (3) Expression of cre in testis of SMMHC-cre mice correlated with 100% efficient rearrangement of tgfbr2flox in male gametes, and germ line transmission of a tgfbr2null allele. In contrast, we found that another line of smooth muscle-targeted cre recombinase mice, SM22α-cre mice,7 efficiently rearranged both tgfbr2flox and R26R in smooth muscle cells. As expected,8 the SM22α-cre X R26R mice also showed significant cre activity in the heart. The primary data that support these conclusions are published elsewhere.4
Recently we became aware that the problem of unanticipated germ line cre expression—though not the problem of low SMC expression—is also found in an independently generated line of SMMHC-cre mice.9 This line shows effective cre-mediated recombination in SMCs but also expresses cre in male germ cells and therefore unexpectedly transmits rearranged floxed alleles (personal communication, Dr Michael Kotlikoff, 2007). Accordingly, when a floxed allele is passed through a SMMHC-cre-positive parent (from either of these two SMMHC-Cre lines),1,9 offspring that would normally receive a floxed allele from that parent instead receive a null allele. These offspring will be heterozygous null throughout whether or not they are cre-positive (see Figure 1C in reference 4). During development, the SMMHC promoter-driven cre is expected to act on the floxed allele inherited from the other parent to convert SMCs to homozygous null. However, because of germ line rearrangement of one floxed allele, the null SMCs that result from somatic SMMHC-Cre activity will be present on a heterozygous null, not a wild-type background. The control littermates will be a mixture of total-body wild-type mice, total-body heterozygous null mice, and mice that are heterozygous null only in SMC (groups 2, 4, and 1, respectively, in Figure 1C in reference 4). Cre-mediated rearrangement of floxed alleles in germ cells can be prevented by passing a null allele instead of a floxed allele through the gametes of the cre-expressing parent. However, the end result will be the same: experimental mice will be heterozygous null throughout and homozygous null in SMCs. The control mice will include the same mixture of genotypes described above. If a heterozygous null genotype in non-SMCs causes a phenotype in the experimental mice (ie, if there is haploinsufficiency), then the effects of total-body haploinsufficiency (present in 100% of experimental mice but only 33% of the littermates) could be mistakenly attributed to SMC-specific gene deletion.
The unanticipated activities of cre recombinase of these two lines of SMMHC-cre mice do not appear to be easy to detect, because in both cases several years elapsed between the time the mice were first reported1,9 and the discovery of their unexpected behavior. It is important that investigators using lines of mice that are designed to express cre only in specific cardiovascular cells recognize the potential for both loss of cre activity and unexpected, seemingly ectopic germ line cre activity. In SMMHC-cre mice, cre expression in gametes might actually be expected based on a report of smooth muscle myosin expression during meiosis.10 An appropriate strategy for minimizing the consequences of unanticipated cre activity is to completely characterize cre-expressing mice soon after their arrival in one’s laboratory and before initiating extensive experimentation that is based on an assumption that the mice will perform as expected. We have described a roadmap for characterizing putative tissue-specific cre mice that we hope will be useful.4
We are grateful to Dr Michael Kotlikoff for generously supplying us with SMMHC-cre mice and for communicating openly with the research community about experimental results obtained with these mice both in his laboratory and elsewhere.
Sources of Funding
Studies referred to in this letter were supported by grants from the NHLBI (HL69063, to D.A.D.) and the John L. Locke Jr Charitable Trust. A.D.F. was supported by NHLBI T32 HL07828 and by the EVEREST foundation.
Regan CP, Manabe I, Owens GK. Development of a smooth muscle–targeted cre recombinase mouse reveals novel insights regarding smooth muscle myosin heavy chain promoter regulation. Circ Res. 2000; 87: 363–369.
Dikalova A, Clempus R, Lassegue B, Cheng G, McCoy J, Dikalov S, San Martin A, Lyle A, Weber DS, Weiss D, Taylor WR, Schmidt HH, Owens GK, Lambeth JD, Griendling KK. Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in transgenic mice. Circulation. 2005; 112: 2668–2676.
Zhang Y, Griendling KK, Dikalova A, Owens GK, Taylor WR. Vascular hypertrophy in angiotensin II-induced hypertension is mediated by vascular smooth muscle cell-derived H2O2. Hypertension. 2005; 46: 732–737.
Leveen P, Larsson J, Ehinger M, Cilio CM, Sundler M, Sjostrand LJ, Holmdahl R, Karlsson S. Induced disruption of the transforming growth factor beta type II receptor gene in mice causes a lethal inflammatory disorder that is transplantable. Blood. 2002; 100: 560–568.
Holtwick R, Gotthardt M, Skryabin B, Steinmetz M, Potthast R, Zetsche B, Hammer RE, Herz J, Kuhn M. Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure. Proc Natl Acad Sci U S A. 2002; 99: 7142–7147.
Miano JM, Ramanan N, Georger MA, de Mesy Bentley KL, Emerson RL, Balza RO Jr, Xiao Q, Weiler H, Ginty DD, Misra RP. Restricted inactivation of serum response factor to the cardiovascular system. Proc Natl Acad Sci U S A. 2004; 101: 17132–17137.
Xin HB, Deng KY, Rishniw M, Ji G, Kotlikoff MI. Smooth muscle expression of Cre recombinase and eGFP in transgenic mice. Physiol Genomics. 2002; 10: 211–215.