Rautureau Yohann, Paradis Pierre, Schiffrin Ernesto L
Lady Davis Institute for Medical Research, McGill University, Montreal, QC, Canada, H3T 1E2.
Lady Davis Institute for Medical Research and Department of Medicine, Jewish General Hospital, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2017;1527:381-407. doi: 10.1007/978-1-4939-6625-7_30.
An inducible tissue-specific knockout (KO) technique has been used to study the role of genes in the adult heart. This KO technique circumvents the developmental effect that could otherwise be observed in a tissue-specific KO. The peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor that when activated has been shown to improve vascular remodeling and endothelial function in hypertensive rodents. Here we describe an inducible tissue specific KO protocol used to study the role of PPARγ in smooth muscle cells (SMC) in angiotensin (Ang) II-induced hypertension in adult mice. Inducible VSMC Pparγ KO mice are generated by crossing mice expressing a fusion protein of Cre recombinase with the modified estrogen receptor ligand binding domain (CreER) under the control of the smooth muscle myosin heavy chain (smmhc, myh11) with mice having loxP sites flanking exon 2 of the Pparγ gene (Pparγ ). The SMC Pparγ KO is induced by treating smMHC-CreER/Pparγ mice with the estrogen receptor antagonist tamoxifen causing recombination of the two loxP site by CreER. SMC KO is confirmed by determining mRNA Pparγ levels in aortic media. Presence of the loxP sites is determined by sequencing genomic DNA. Tissue specific expression is assayed using smMHC-CreER/reporter crossed mice.
一种可诱导的组织特异性敲除（KO）技术已被用于研究基因在成年心脏中的作用。这种KO技术规避了在组织特异性KO中可能观察到的发育效应。过氧化物酶体增殖物激活受体（PPAR）γ是一种转录因子，在高血压啮齿动物中，其激活后已被证明可改善血管重塑和内皮功能。在此，我们描述了一种可诱导的组织特异性KO方案，用于研究PPARγ在成年小鼠血管紧张素（Ang）II诱导的高血压中平滑肌细胞（SMC）中的作用。可诱导的血管平滑肌细胞Pparγ KO小鼠是通过将在平滑肌肌球蛋白重链（smmhc，myh11）控制下表达Cre重组酶与修饰的雌激素受体配体结合域（CreER）融合蛋白的小鼠与Pparγ基因（Pparγ ）外显子2两侧带有loxP位点的小鼠杂交而产生的。通过用雌激素受体拮抗剂他莫昔芬处理sMMHC-CreER/Pparγ 小鼠，使CreER介导两个loxP位点发生重组，从而诱导SMC Pparγ KO。通过测定主动脉中膜的mRNA Pparγ水平来确认SMC KO。通过对基因组DNA进行测序来确定loxP位点的存在。使用sMMHC-CreER/报告基因杂交小鼠来检测组织特异性表达。
