Transgenic and Knockout Models of ADPKD

我们以前开发的转基因株系，表达人类常染色体显性遗传性多囊肾病基因，PKD 1，从一个大的基因组片段（TPK）。 转基因挽救了Pkd 1-/-小鼠的致死表型，但TPK动物经常发展为肾脏和肝脏囊性疾病。 这些结果表明，PKD 1蛋白，多囊蛋白-1的水平，可能是重要的维持正常的肾脏结构。 本提案的第一部分是通过产生含有PKD 1的较小基因组插入物的PKD 1转基因系来澄清这些发现，但不是相邻结节性硬化症基因TSC 2的转录活性拷贝，如在原始TPK动物中存在的那样。 这些转基因小鼠将阐明TSC 2基因对于PKD 1正常表达的重要性。 此外，他们将显示是否过度表达多囊蛋白-1足以导致囊性表型。 随后，将制备具有精确定义的功能性PKD 1拷贝数的转基因动物。 将比较具有不同PKD 1表达水平的动物的表型结果和拯救潜力。 还将评估具有截短或基序特异性框内或错义突变的突变PKD 1基因的表达模式、稳定性、表型后果和拯救潜力。 这些实验将测试截短的多囊蛋白-1分子的显性负电位。此外，通过检查与Pkd 1-/-小鼠获救，或部分获救，由基序特异性突变的转基因相关的表型，单个多囊蛋白-1域的作用将被阐明。 该提案的第二部分将通过建立Pkd 1的条件性敲除来测试多囊蛋白-1在新生儿和成人生活中的功能。 内源性鼠Pkd 1将通过插入外显子1侧翼的LoxP位点进行修饰。在与含有在Tet-On系统控制下的Cre重组酶基因的转基因小鼠杂交后，通过添加多西环素来诱导产生无效等位基因Pkd 1dell的突变。 Cre表达将在新生儿或成年期间被激活短时间，以检查存活动物中无效多囊蛋白-1细胞的命运。 通过将Tet-On系统置于仅在肾中表达的组织特异性启动子（例如Ksp-钙粘蛋白）的控制下，Cre重组将进一步导向特定的器官和/或组织。 该系统将允许Pkd 1失活的时间和空间控制，并允许在不同器官和细胞类型中研究多囊蛋白-1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.