肾脏特异性表达Cre重组酶工具猪的鉴定与品系的建立

Kidney disease has serious impact on human health and life quality. Ideal animal model will bring new breakthrough for the research and treatment of kidney disease. At present, although some mice kidney disease models have been set up, not all of them are suitable for the research of human disease. Miniature Pig is an ideal experimental animal for the study of human disease because its structure and physiological characteristics are similar to human beings. In order to establish a kidney disease model with conditional knock out of associated genes,first of all need to establish tissue-specific expression of Cre recombinase transgenic pigs. AQP2-Cre miniature pig was constructed by us, this research will aim at the identification of AQP2-Cre pig, and analyzing the Cre recombinase activity through the hybridation with the former Cre report pig. At last, the number of this kind of genetically modified pig will be enlarged to cultivate it as a stable strain. This research will lay the foundation for using conditional knockout techniques to study the specific gene in the kidney development and kidney disease.

肾脏疾病是严重影响人类生命健康及生活质量的疾病。理想动物模型的建立将为其研究与治疗带来新的突破。目前已经建立一些小鼠的肾脏疾病模型，但有些模型不适合用于人类疾病的研究。结构与生理特性与人类极为相似的小型猪是研究人类疾病的理想实验动物。为了建立条件性基因敲除的肾脏疾病模型，首先需要建立组织特异性表达Cre重组酶的转基因猪。本研究在前期获得的AQP2-Cre小型猪的基础上，对AQP2-Cre克隆猪进行鉴定，并与前期获得的 Cre报告猪杂交，检测Cre重组酶活性；将肾脏特异性表达且具有Cre重组酶活性的转基因猪扩繁，培养成稳定品系。为利用条件性基因敲除技术，研究特定基因在肾脏发育及肾脏相关疾病中的功能奠定基础。

肾脏疾病是严重影响人类生命健康及生活质量的疾病。为了建立适合人类肾脏疾病研究的小型猪的动物模型，我们选择猪源 AQP2基因启动子区序列来驱动 Cre 重组酶表达。本研究利用3,090 bp猪源AQP2转录起始位点上游序列驱动Cre重组酶表达的表达载体，来构建肾脏特异性表达Cre重组酶的转基因克隆猪，并获得12头克隆猪，通过检测，结果表明转基因小型猪基因组整合了完整的 Cre 表达序列，分析转基因小型猪 Cre 表达情况。检测结果显示，Cre 重组酶表达于AQP2-Cre 转基因小型猪肺脏组织，其它组织中不表达。免疫组化分析结果表明，Cre 重组酶表达于肺泡上皮细胞，且细胞质与细胞核均有表达。将猪源AQP2基因启动子区序列由3090bp增加到8000bp，驱动Cre重组酶表达，构建转基因克隆猪，经过11次核移植，共生产 9头克隆小型猪,2头存活。检测结果表明，Cre重组酶只在肾脏中特异性表达。AQP2-Cre 克隆猪的2464号进行了杂交，生产后，分离F1代各个组织，鉴定Cre重组酶的活性及分布。并以获得的小型猪为基础进行扩繁。