GENETIC TRANSFORMATION OF RAT EMBRYOS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 背景和目的：在大鼠和人脑中已经证实了合成醛固酮所需的所有酶的表达和活性。为了研究中枢合成的醛固酮在高血压和靶器官损伤中的潜在作用，我们建立了一种在神经元中选择性过度表达醛固酮合成酶(AS)的转基因大鼠(SYN1/AS-TG大鼠)。方法：利用慢病毒载体技术，在大鼠突触素-1启动子(4.5kbp)的控制下过表达大鼠醛固酮合成酶基因，以克服传统的大鼠胚胎注射裸DNA转基因方法的局限性。结果：实时定量RT-PCR检测到SYN1/AS-TG大鼠脑内AS基因表达水平是WT大鼠(n=8)的100倍。在标准大鼠饲料中，0.28%食盐的SYN1/AS-TG的血压显著高于清醒自由活动大鼠的平均动脉压，分别为20 mm Hg(n=11)和4(n=4)。SYN1/AS-TG大鼠与WT大鼠(n=5)相比，尿醛固酮水平无明显差异，说明转基因大鼠在全身RAS调控下所产生的醛固酮水平并未超过肾上腺所产生的水平，且中枢水平升高可导致SYN1/AS-TG大鼠血压升高。在13周龄时，与WT对照组相比，SYN1/AS-TG组有明显的心肌肥大、蛋白尿、肾脏胶原和转化生长因子-β。被允许存活超过12个月的少数动物在14个月大时出现了严重的充血性心力衰竭。 结论：这种新颖而独特的大鼠模型将使我们能够研究神经元中较高水平的醛固酮对心血管系统的全身性影响，而不是外周器官中醛固酮过量的直接影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background and Aims: The expression and activity of all of the enzymes required for aldosterone synthesis have been demonstrated in the rat and human brain. To study the potential role of centrally synthesized aldosterone in hypertension and target organ damage, we developed a transgenic rat (Syn1/AS-Tg rat) that over-expresses aldosterone synthase (AS) selectively in neurons. Methods: We generated our transgenic rat model by over-expressing the rat aldosterone synthase cDNA under the control of the rat synapsin-1 promoter (4.5 kbp) using lentiviral delivery to overcome the limitations of traditional transgenesis by naked DNA injection in rat embryos. Results: AS mRNA in the brain measured by real time RT-PCR was 100 times greater in Syn1/AS-Tg rats compared with WT rats (n=8). The blood pressure of the Syn1/AS-Tg on a standard rat chow, 0.28% salt, had significantly higher, 20 mmHg, mean arterial pressures both by tail cuff(n=11) and by indwelling catheter (n=4) in the conscious freely moving rat. Urinary aldosterone, an index of circulating aldosterone, was not different in Syn1/AS-Tg compared with WT rats (n=5), indicating that the amount of aldosterone produced by the transgene did not exceed that produced by the adrenal under control of the systemic RAS and that central increases in aldosterone alone lead to increased blood pressure in the Syn1/AS-Tg. At 13 weeks of age the Syn1/AS-Tg had significant cardiac hypertrophy, proteinuria, and renal collagen and TGF-beta as compared to WT controls. The few animals allowed to live past 12 months of age experienced severe congestive heart failure by 14 months of age. Conclusion: This novel and unique rat model will allow us to study the systemic effects of higher levels of aldosterone in neurons on the cardiovascular system without the direct of effects of aldosterone excess in peripheral organs.