Stec D E, Davisson R L, Haskell R E, Davidson B L, Sigmund C D
Departments of Internal Medicine and Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 1999 Jul 23;274(30):21285-90. doi: 10.1074/jbc.274.30.21285.
Tissue-specific ablation of gene function is possible in vivo by the Cre-loxP recombinase system. We generated transgenic mice containing a human angiotensinogen gene flanked by loxP sites (hAGT(flox)). To examine the physiologic consequences of tissue-specific loss of angiotensinogen gene function in vivo, we constructed an adenovirus expressing Cre recombinase. Studies were performed in several independent lines of hAGT(flox) mice before and after intravenous administration of either Adcre or AdbetaGal as a control. Systemic administration of Adcre caused a significant decrease in circulating human angiotensinogen and markedly blunted the pressor response to administration of purified recombinant human renin. Southern blot analysis of genomic DNA from various organs revealed that the Cre-mediated deletion was liver-specific. Further analysis revealed the absence of full-length human angiotensinogen mRNA and protein in the liver but not the kidney of Adcre mice, consistent with the liver being the target for adenoviruses administered intravenously. These studies demonstrate that extra-hepatic sources of angiotensinogen do not contribute significantly to the circulating pool of angiotensinogen and provide proof-of-principle that the Cre-loxP system can be used effectively to examine the contribution of the systemic and tissue renin-angiotensin system to normal and pathological regulation of blood pressure.
通过Cre-loxP重组酶系统可在体内实现基因功能的组织特异性缺失。我们构建了转基因小鼠，其携带loxP位点侧翼的人血管紧张素原基因（hAGT(flox)）。为了在体内研究血管紧张素原基因功能的组织特异性缺失的生理后果，我们构建了一种表达Cre重组酶的腺病毒。在静脉注射Adcre或作为对照的AdbetaGal之前和之后，对多个独立品系的hAGT(flox)小鼠进行了研究。全身给予Adcre导致循环中的人血管紧张素原显著减少，并明显减弱了对纯化重组人肾素给药的升压反应。对来自各种器官的基因组DNA进行Southern印迹分析表明，Cre介导的缺失是肝脏特异性的。进一步分析显示，Adcre小鼠的肝脏中不存在全长人血管紧张素原mRNA和蛋白质，但肾脏中存在，这与肝脏是静脉注射腺病毒的靶器官一致。这些研究表明，血管紧张素原的肝外来源对循环中的血管紧张素原池贡献不大，并提供了原理证明，即Cre-loxP系统可有效用于研究全身和组织肾素-血管紧张素系统对血压正常和病理调节的贡献。
