Pumin Zhang Group (GUDMAP1)
The development of genitourinary tract, like any other organ systems, is very complex that requires controlled proliferation and differentiation of various types of cells. To understand the developmental processes that lead to the formation of the GU tract, we need genetic tools that will enable us to monitor gene expression dynamics in vivo, to dissect cell-cell interactions, to trace cell lineages, and to perform biochemical analyses. Our GUDMAP project focuses on generating such tools. We are genetically marking or labeling the different types of cells in the mouse GU tract through knocking fluorescent protein markers and/or the Cre recombinase, or epitope tags, into genes which are specifically expressed in those cell types.
We are taking the gene targeting approach in mouse embryonic stem cells. First, a targeting vector is constructed in which the selected marker plus a drug resistance gene-expressing cassette is flanked by sequences homologous to the locus being modified. Second, the vector is introduced into ES cells and drug-resistant ES clones are isolated. Third, the ES clones are screened for homologous recombinants. Fourth, mice carrying the modified gene loci are derived from the ES clones and used in analyses. The ES clones and/or the mouse strains, once generated, are available freely to the research community.
We are using homologous recombination-based methods or recombineering (http://recombineering.ncifcrf.gov/) for the construction of the targeting vectors. Three types of vectors are generated in which different combinations of expression cassettes are inserted at the starting codons of the genes of interest. A is for the regulable expression of the GFP::Cre fusion product. The expression of tTA is controlled by the targeted locus, which then controls the expression of GFP::Cre via the tetO-CMVmin promoter. The activity of tTA can be regulated by the presence or absence of tetracycline. B is used to express the red fluorescent protein from an endogenous locus. C is used to first express the TAP::GFP fusion product for monitoring the dynamics of gene expression. TAP stands for tandom-affinity-purification tag. It is composed of protein A IgG binding domain, TEV protease site, and triple FLAG tag. After Cre-mediated loopout, TAP is fused to the endogenous gene product, making possible the biochemical purification and characterization of the protein complexes containing the targeted gene product.