Core B: Engineered Models Resource Core
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One of the primary objectives of the Engineered Models Core is to develop and disseminate important mouse resources related to cystic kidney disorders. The core continues to build important mouse resources with six new conditional mutant lines in production this funding period. These include three genes known to cause Meckel-Gruber Syndrome (MKS) and one involved in Nephronophthisis (NPHP). Conditional alleles for these genes are not currently available from any other source. Since many of these mutants are generally lethal during early development it is not possible to analyze the role in later development or in postnatal stages without conditional alleles. All together, the core has established 24 different mouse mutants in cilia related genes and has generated several of the corresponding embryonic fibroblast or renal cell lines for in vitro analysis. The initial characterization of the phenotype and genotyping strategies for most lines has been established (see table) and the mice/cell lines are available for Center members to use in their studies.
In the second aim, the goal is to establish an efficient knock-in approach for uniform Cre-induced expression of HRFD related genes. This will allow in vivo analysis of gene function, localization, interactions, etc. The core has received numerous requests for this service and in the past year we have initiated six independent projects for center members at UAB. As a demonstration of its utility to HRFD and cilia biology related research, the initial project started by the core was to generate an inducible cilia tagged mouse (please see this PowerPoint presentation along with the two movies: Core B - Cilia Tag Mouse.ppt; Sham tubules.avi; Ventricle cilia.avi). For this we cloned the somatostatin receptor 3 (SSTR3) cDNA fused to GFP into a Gateway modified Rosa26 targeting vector. This was correctly recombined into the Rosa26 locus in about 25% of the clones screened. To test the system prior to making the mouse line, the ES cells were infected with a Cre virus and differentiated to allow cilia to form. In all clones in which correct Rosa26 targeting occurred, GFP tagged cilia were evident. We have generated the chimeras from the targeted ES cells and have recently obtained the first mice with germline transmission of the SSTR3-GFP. These mice are being crossed with Cre lines to test their utility for visualizing cilia in vivo in living samples.
The overall goal of the third aim is to provide opportunity for center members to utilize C. elegans in their analysis of HRFD genes. This includes assessing protein localization, function, and potential genetic interactions with mutations in other ciliopathy genes. The core has developed numerous resources that are available to center members that include mutations in many of the ciliopathy genes as well as transgenic animals expressing the ciliopathy proteins fused with various fluorophores.
Core Services and Resources:
) to request resources or to make suggestions for new mouse/C. elegans/cell lines.
Publications with assistance of the core