Transgenic and Knockout Models of ADPKD

我们之前已经开发出了从大基因组片段（TPK）中表达人类常染色体显性多囊肾病基因PKD1的转基因系。转基因挽救了Pkd1-/-小鼠的致死表型，但TPK动物经常出现肾脏和肝脏囊性疾病。这些结果表明PKD1蛋白多囊蛋白-1的水平可能对维持正常的肾脏结构很重要。该提案的第一部分是通过产生PKD1转基因系来澄清这些发现，这些系含有较小的基因组插入物，其中包含PKD1，但不像原始TPK动物中存在的相邻结节性硬化症基因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，他们将显示是否过度表达多囊蛋白-1足以引起囊性表型。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。比较不同PKD1表达水平的动物的表型后果和救援潜力。还将评估突变PKD1基因的表达模式、稳定性、表型后果和挽救潜力，包括截断或基序特异性框架内或错义突变。这些实验将测试截短的多囊蛋白-1分子的主要负电位。此外，通过检测与带有基序特异性突变的转基因获救或部分获救的Pkd1-/-小鼠相关的表型，将阐明单个多囊蛋白-1结构域的作用。该提案的第二部分将通过创建Pkd1的条件敲除来测试多囊蛋白-1在新生儿和成人生活中的功能。内源性小鼠Pkd1将通过插入1号外显子两侧的LoxP位点进行修饰。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，加入强力霉素诱导重组产生空的Pkd1dell等位基因。Cre表达将在新生期或成年期被激活一段短时间，以检测存活动物中无效多囊蛋白-1细胞的命运。通过将Tet-On系统置于组织特异性启动子（如仅在肾脏中表达的ksp -钙粘蛋白）的控制下，Cre重组将进一步定向到特定的器官和/或组织。该系统将允许对Pkd1失活的时间和空间控制，并允许多囊蛋白-1在不同器官和细胞类型中的发育后作用进行研究。

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.