Transgenic and Knockout Models of ADPKD

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date4/1/011/31/17

ASJC

  • Medicine(all)