Gene recombination in postmitotic cells - Targeted expression of cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo
Gene recombination in postmitotic cells - Targeted expression of cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo
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DOI：
10.1172/jci119509
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发表时间：
1997-07-01
影响因子：
15.9
通讯作者：
Schneider, MD
中科院分区：
文献类型：
--
作者：
Agah, R;Frenkel, PA;Schneider, MD
人类疾病的小鼠模型可以通过胚系功能丧失突变的同源重组来产生，然而，胚胎致死表型和全身性、间接功能障碍可能会混淆利用敲除来阐明成人的病理生理学，使用Cre重组酶的位置特异性重组可以绕过这些陷阱，原则上，能够实现对基因重组的时间和空间控制。然而，对于Cre介导的重组在有丝分裂后细胞中的可行性缺乏直接的证据，在这里，我们利用转基因小鼠技术和腺病毒基因转移来实现Cre介导的心肌重组。在体外，由心脏特异的α-肌球蛋白重链(αMyHC)序列驱动的Cre在纯化的心肌细胞中选择性地在loxP位点引起重组，但不能在心脏成纤维细胞中诱导重组。在体内，这种α-MyHC-Cre转基因引起了心肌中的重组，但不能引起其他器官的重组。通过PCR分析和重组依赖的报告蛋白的定位，腺病毒体内递送Cre在体内引起了有丝分裂后的成年心室肌细胞的重组。在没有Cre的情况下，没有检测到loxP位点之间的重组。这些研究证明了使用Cre介导的重组来调控心肌中基因表达的可行性，即使在终末分化的有丝分裂后的肌细胞中也能有效地诱导重组，此外，通过病毒感染传递Cre为控制心肌中重组的时间提供了一种简单的策略。
Mouse models of human disease can be generated by homologous recombination for germline loss-of-function mutations, However, embryonic-lethal phenotypes and systemic, indirect dysfunction can confound the use of knock-outs to elucidate adult pathophysiology, Site-specific recombination using Cre recombinase can circumvent these pitfalls, in principle, enabling temporal and spatial control of gene recombination. However, direct evidence is lacking for the feasibility of Cre-mediated recombination in postmitotic cells, Here, we exploited transgenic mouse technology plus adenoviral gene transfer to achieve Cre-mediated recombination in cardiac muscle. In vitro, Cre driven by cardiac-specific alpha-myosin heavy chain (alpha MyHC) sequences elicited recombination selectively at loxP sites in purified cardiac myocytes, but not cardiac fibroblasts. In vivo, this alpha MyHC-Cre transgene elicited recombination in cardiac muscle, but not other organs, as ascertained by PCR analysis and localization of a recombination-dependent reporter protein, Adenoviral delivery of Cre in vivo provoked recombination in postmitotic, adult ventricular myocytes. Recombination between loxP sites was not detected in the absence of Cre. These studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells, Moreover, delivery of Cre by viral infection provides a simple strategy to control the timing of recombination in myocardium.