Temporally controlled somatic mutagenesis in smooth muscle

Abstract

Ligand-dependent site-specific recombinases are powerful tools to engineer the mouse genome in specific somatic cell types at selected times during pre- and postnatal development. Current efforts are primarily directed towards increasing the efficiency of this recombination system in mice. We have generated transgenic mouse lines expressing a tamoxifen-activated Cre recombinase, CreER(T2), under the control of the smooth muscle-specific SM22 promoter. Both a randomly integrated transgene [SM-CreER(T2)(tg)] and a transgene that has been "knocked in" into the endogenous SM22 locus [SM-CreER(T2)(ki)] were expressed in smooth muscle-containing tissues. The level of CreER(T2) expression and tamoxifen-induced recombination was lower in SM-CreER(T2)(tg) mice compared with SM-CreER(T2)(ki) mice. Whereas no recombinase activity could be detected in vehicle-treated SM-CreER(T2)(ki) mice, administration of tamoxifen induced the excision of a loxP-flanked reporter transgene in up to 100% of smooth muscle cells. The recombined genome persisted for at least four months after tamoxifen treatment. SM-CreER(T2)(ki) transgenic mice should be useful to study the effects of various somatic mutations in smooth muscle.

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death

“…To this end, conditional inactivation of both genes was carried out in mice harboring mdm2 or mdm4 floxed alleles and a tamoxifen (TMX)-inducible Cre-recombinase under the control of the SM22 promoter (SM-CreER T2 (ki) mice). 23 The mdm2 and the mdm4 floxed alleles (FM) had been previously described. 24,25 They carry loxP recombination sites in introns 4 and 6 of mdm2 and in introns 1 and 2 of mdm4 (Figure 1a).…”

“…To achieve SMC-specific mdm2 and mdm4 deletion, we combined mice that carry a TMX-inducible Cre-recombinase under the control of the SMCspecific SM22 promoter (SM-CreER T2 (ki) mice) 23 and mice carrying mdm2 or mdm4 floxed alleles 24,25 to create SM-CreER T2 (ki);mdm2 FM/FM and CreER T2 (ki);mdm4 FM/FM mice. To determine p53-dependent effects of mdm2 deletion, SM-CreER T2 (ki);mdm2 FM/FM mice were crossed with p53 knockout (p53 À/À ) mice 27 resulting in SM-CreER T2 (ki);mdm2 FM/FM p53 À/À mice.…”

Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system

“…Again, in our experience, intermediate doses are generally well tolerated in Swiss Webster female mice, giving efficient recombination and good litter viability. Oral gavage delivery of tamoxifen appears to reduce litter loss apparently associated with intraperitoneal inflammation resulting from direct injection (unpublished observation), with both methods providing similar recombination efficiency (Kuhbandner et al, 2000). Progesterone (approximately 1.5-2.0 mg/40 g pregnant female) also appears to improve litter viability when giving high doses of tamoxifen (unpublished observation).…”

“…4-hydroxytamoxifen is a metabolite of tamoxifen that is generated in the liver and believed to be the active compound in binding the ER, although administration of either compound, at the same concentration, results in indistinguishable recombination efficiency (Kuhbandner et al, 2000). Importantly, embryonic stages are particularly sensitive to high tamoxifen doses and may result in lethality, especially in inbred strains.…”

Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

“…for five consecutive days at a dose of 1mg per day as described. 15 The tamoxifen (100 mg, Sigma, T5648) was dissolved in 0.5 ml ethanol followed by 9.5 ml sunflower oil at a concentration of 10 mg/ml and stored at −20°C for up to one month. All mice used in this study were of a mixed 129/B6 background.…”