Transgenic Organ Cultures

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DESCRIPTION (provided by applicant): Our knowledge of the molecular basis for kidney development has expanded dramatically over the last 10 years. Much of this new information has come from the study of transgenic and gene-targeted mice. However, there are serious limitations to this experimental approach. First, there is the cost and time involved in the production of each strain of knockout mouse. Secondly, taking an example from our own work with the Wtl gene, the early demise of the metanephric rudiment leaves little actual material to study in order to gain further insight as to the mechanism by which Wt1 is involved in early kidney development [3, 4]. Further study of the Wt1 gene in tissue culture settings has added little to our understanding of its role in early kidney development [5]. Because of these limitations, we have sought to develop alternative and less costly ways of studying the molecular basis of early kidney development. Recently, several studies have demonstrated that it is possible to use electroporation to achieve gene transfer into embryonic tissues; this has found particular use in the study of neural tube and limb development [6-10]. In our laboratory, we are adapting electroporation technology to achieve gene transfer into metanephric kidney organ cultures. In contrast to work with strains of mutant mice, several advantages are immediately apparent. (1) there can be much higher throughput in studying the effects of specific genes on kidney development; (2) wild type organ cultures are used, hence every embryo provides 2 organ cultures, instead of the 25 % of embryos from a litter that might be homozygous for a mutation; (3) in cases like Wt1, where the knockout causes apoptosis, alternate experimental approaches are available, for example those that examine gain of function phenotypes. Microinjection and electroporation will be used to elucidate the role of WT1 in early kidney development. Hypotheses relating to whether Wtl regulates various growth factors will be tested by ectopically expressing or inhibiting Wt1 in the metanephric mesenchyme, and examining the expression of potential target genes by in situ hyridization.