Transgenic and Knockout Models of ADPKD

我们之前已经从一个大的基因组片段(TPK)中开发出表达人类常染色体显性遗传性多囊肾病基因PKD1的转基因株。转基因可以挽救PKD-1-/-小鼠的致死性表型，但TPK-/-小鼠通常会发生肾和肝囊性疾病。这些结果表明，多囊蛋白-1的水平可能是维持正常肾脏结构的重要因素。这项建议的第一部分是通过建立含有PKD1的较小基因组插入的PKD1转基因系来澄清这些发现，而不是像最初的TPK动物中存在的那样，具有邻近结节性硬化症基因TSC2的转录活性拷贝。这些转基因小鼠将阐明TSC2基因对PKD1正常表达的重要性。此外，他们还将表明，仅仅过度表达多囊蛋白-1是否足以导致囊性表型。随后，将制备具有精确定义的功能性PKD1拷贝数的转基因动物。将比较具有不同水平PKD1表达的动物的表型后果和拯救潜力。还将评估突变的PKD1基因的表达模式、稳定性、表型后果和拯救潜力，这些基因带有截断或基序特异的框内或错义突变。这些实验将测试截短的多囊蛋白-1分子的显性负势。此外，通过检测通过基序特异突变的转基因拯救或部分挽救的PKD1-/-小鼠的表型，将阐明单个多囊蛋白-1结构域的作用。提案的第二部分将通过建立有条件的蛋白激酶1基因敲除来测试多囊蛋白-1在新生儿和成人生活中的功能(S)。通过在外显子1两侧插入loxP位点来修饰内源性小鼠的PKD1。在Tet-on系统的控制下，与含有Cre重组酶基因的转基因小鼠杂交后，加入强力霉素将诱导重组产生空的Pkd1dell等位基因。Cre的表达将在新生儿或成年生命的短时间内被激活，以检查活着的动物体内无效的多囊蛋白-1细胞的命运。通过将Tet-on系统置于组织特异性启动子的控制下，Cre重组将进一步定向到特定的器官和/或组织，例如只在肾脏表达的KSP-cadherin。该系统将允许对PKD1失活的时间和空间进行控制，并允许研究多囊蛋白-1在不同器官和细胞类型中的发育后作用。

We have previously developed transgenic lines that express the human autosomal dominant polycystic kidney disease gene, PKD1, from a large genomic fragment (TPK). The transgene rescues the lethal phenotype of Pkd1-/- mice, but the TPK animals often develop renal and hepatic cystic disease. These results indicated that the level of PKD1 protein, polycystin-1, may be important for maintaining normal renal architecture. The first part of this proposal is to clarify these findings by generating PKD1 transgenic lines with a smaller genomic insert containing PKD1, but not a transcriptionally active copy of the adjacent tuberous sclerosis gene, TSC2, as was present in the original TPK animals. These transgenic mice will clarify the importance of the TSC2 gene for normal expression of PKD1. Furthermore, they will show whether overexpressing just polycystin-1 is sufficient to cause a cystic phenotype. Subsequently, transgenic animals will be prepared with precisely defined copy numbers of functional PKD1. The phenotypic consequences and rescue potential of animals with different levels of PKD1 expression will be compared. The expression pattern, stability, phenotypic consequences and rescue potential of mutant PKD1 genes, with truncating, or motif specific in-frame or missense mutations, will also be assessed. These experiments will test the dominant negative potential of truncated polycystin-1 molecules. Furthermore, by examining the phenotypes associated with Pkd1-/- mice rescued, or partially rescued, by transgenes with motif specific mutations, the role of individual polycystin-1 domains will be elucidated. The second part of the proposal will test the function(s) of polycystin-1 during neonatal and adult life by creating conditional knockouts of Pkd1. The endogenous murine Pkd1 will be modified by insertion of LoxP sites flanking exon 1. Recombination to generate a null, Pkd1dell, allele will be induced by the addition of doxycycline after crossing with a transgenic mouse containing a Cre recombinase gene under the control of the Tet-On system. Cre expression will be activated for short periods during neonatal or adult life to examine the fate of null polycystin-1 cells in a viable animal. Cre recombination will further be directed to specific organs and/or tissues by placing the Tet-On system under the control of a tissue specific promoter, such as Ksp-cadherin, that is only expressed in the kidney. This system will allow temporal and spatial control of Pkd1 inactivation and allow the post- developmental role of polycystin-1 to be investigated in different organs and cell types.