Regan C P, Manabe I, Owens G K
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA.
Circ Res. 2000 Sep 1;87(5):363-9. doi: 10.1161/01.res.87.5.363.
The use of genetically modified mice has been an important model system to study gene function in cardiovascular development and under pathophysiological conditions. Although conventional gene knockout studies have provided important insights into gene function in the cardiovascular system, they may be limited by upregulation of compensatory pathways and the inability to differentiate direct versus indirect functions in vivo. As a first step in developing systems that can target gene activation or inactivation specifically to smooth muscle cells (SMCs), we coupled the smooth muscle myosin heavy chain (SMMHC) promoter to the cre recombinase gene and generated transgenic mice that express cre in SMCs. In addition, we used these mice to address whether the heterogeneous staining observed in SMMHC-LacZ mice was due to subsets of SMCs that required different regulatory cassettes of the promoter or if it reflected episodic expression of the transgene. To address both the feasibility of SMC targeting and the apparent heterogeneous expression, we bred SMMHC-cre mice to indicator mice containing a cre-activated LacZ gene. Results showed high-level expression in SMCs at various embryonic time points and in adult tissues. Because breeding of SMMHC-cre mice to an indicator line provided an integration of cre activity over time, results of this study revealed that expression of the SMMHC promoter fragment more closely resembled the expression of the endogenous gene, both with respect to the onset of activation during development and uniformity of staining among individual cells within tissues. Overall, these mice will 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 in vivo.
转基因小鼠的使用已成为研究心血管发育和病理生理条件下基因功能的重要模型系统。尽管传统的基因敲除研究为心血管系统中的基因功能提供了重要见解，但它们可能受到代偿途径上调以及无法在体内区分直接与间接功能的限制。作为开发能够将基因激活或失活特异性靶向平滑肌细胞（SMC）的系统的第一步，我们将平滑肌肌球蛋白重链（SMMHC）启动子与cre重组酶基因偶联，并生成了在SMC中表达cre的转基因小鼠。此外，我们利用这些小鼠来探讨在SMMHC-LacZ小鼠中观察到的异质性染色是由于需要启动子不同调控元件的SMC亚群所致，还是反映了转基因的间歇性表达。为了探讨SMC靶向的可行性和明显的异质性表达，我们将SMMHC-cre小鼠与含有cre激活的LacZ基因的指示小鼠进行杂交。结果显示在不同胚胎时间点的SMC以及成年组织中均有高水平表达。由于将SMMHC-cre小鼠与指示系杂交可随时间整合cre活性，本研究结果表明，SMMHC启动子片段的表达在发育过程中的激活起始以及组织内单个细胞间染色的均匀性方面，都更类似于内源性基因的表达。总体而言，这些小鼠将为研究人员提供一个强大的工具，通过使用cre/lox技术在体内直接进行平滑肌特异性基因激活或失活，来研究血管发育/疾病中的基因功能。
