TARGETED KNOCKOUT OF PKD1 GENE IN RENAL COLLECTING DUCT

描述(改编自申请人的摘要)：对 PKD1在肾脏结构的发展和维持中的作用将是 通过建立具有PKD1基因突变的动物模型而取得进展 肾囊肿并存活到成年期。PKD1外显子34缺失小鼠发育为肾脏 包囊，但会在围产期死亡。这项提议旨在培育一种动物 PKD1基因突变致肾功能受限的ADPKD模型 形成包囊，不影响其他器官。Cre/loxP重组 将利用该系统选择性地截断肾脏中的PKD-1基因 含人水通道蛋白-2启动子的集合管细胞表达 标记Cre重组酶的表位。表达Cre的转基因小鼠 选择性地在肾脏集合管中已制备。基因打靶 技术将被用来创造纯合子的小鼠的牙线外显子34 小鼠PKD1基因。这些小鼠将与AQP2-CreTag小鼠交配产生 AQP2-CreTag杂合子和AQP2-CreTag杂合子或纯合子小鼠 将PKD_1基因转化为FLOCK。我们预测CreTag只在 收集管将切除已脱落的PKD1外显子34。由此产生的突变体PKD1 转录产物应被截短，并表达无功能的PKD1蛋白 有选择地在收集管道中。表型将被确定，预期 该囊肿只会在髓质收集管中形成。将使用RT-PCR 为了鉴定突变型PKD1mRNA，Polycstin的表达缺失 免疫组织化学和组织学技术用于分析囊性病变 队形。我们预测携带集合管特异性PKD1基因的动物 截肢会发展成肾囊肿，但仍能存活到成年。这只动物 模型可以用来研究与人类有关的囊性发生和治疗方法 ADPKD.

DESCRIPTION (Adapted from the Applicant's Abstract): An understanding of the role of PKD1 in the development and maintenance of renal architecture would be advanced by developing an animal model with a PKD1 gene mutation that develop renal cysts and survives into adulthood. PKD1 exon 34 null mice develop renal cysts but die in the perinatal period. This proposal aims to develop an animal model of ADPKD with a mutation in the PKD1 gene that results in limited renal cyst formation and does not effect other organs. The Cre/loxP recombination system will be utilized to selectively truncate the PKD-1 gene in renal collecting duct cells with the human aquaporin-2 promoter driving expression of an epitope tagged Cre recombinase. Transgenic mice in which Cre is expressed selectively in the renal collecting duct have been prepared. Gene targeting techniques will be used to create mice homozygous for floxed exon 34 of the murine PKD1 gene. These mice will be mated with AQP2-CreTag mice to produce mice heterozygous for AQP2-CreTag and either heterozygous or homozygous for the floxed PKD1 gene. We predict that CreTag expressed exclusively in the collecting duct will excise floxed PKD1 exon 34. The resulting mutant PKD1 transcript should be truncated, and non-functional PKD1 protein expressed selectively in the collecting duct. Phenotype will be determined, anticipating that cysts will only form in the medullary collecting duct. RT-PCR will be used to identify mutant PKD1 mRNA, loss of polycstin expression demonstrated by immunohistochemistry, and histological techniques employed to analyze cyst formation. We predict that animals harboring collecting duct specific PKD1 gene truncation will develop renal cysts, yet survive into adulthood. This animal model could then be used to study cystogenesis and therapies relevant to human ADPKD.