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Rapid Isolation of Glomeruli Coupled with Gene Expression Profiling Identifies Downstream Targets in Pod1 Knockout Mice

Shiying Cui^*, Chengjin Li^*, Masatsugu Ema, Jordan Weinstein and Susan E. Quaggin^*,

^* The Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada; Department of Anatomy and Embryology, Institute of Basic Medical Sciences, Division of Developmental Technology, Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki, Japan; and Division of Nephrology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada

Address correspondence to: Dr. Susan E. Quaggin, The Samuel Lunenfeld Research Institute, Department of Maternal Health & Fetal Health, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada. Phone: 416-586-4800 ext. 2859; Fax: 416-586-8588; quaggin{at}mshri.on.ca

Received for publication March 14, 2005. Accepted for publication August 4, 2005.

Mouse mutations have provided tremendous insights into the molecular basis of renal and glomerular development. However, genes often play important roles during multiple stages of nephrogenesis, making it difficult to determine the role of a gene in a specific cell lineage such as the podocyte. Conditional gene targeting and chimeric analysis are two possible approaches to dissect the function of genes in specific cell populations. However, these are labor-intensive and costly and require the generation, validation, and analysis of additional transgenic lines. For overcoming these shortcomings and, specifically, for studying the role of gene function in developing glomeruli, a technique to isolate and purify glomeruli from murine embryos was developed. Combined with gene expression profiling, this method was used to identify differentially expressed genes in glomeruli from Pod1 knockout (KO) mice that die in the perinatal period with multiple renal defects. Glomeruli from early developing stages (late S-shape/early capillary loop) onward can be isolated successfully from wild-type and KO kidneys at 18.5 d postcoitus, and RNA can readily be obtained and used for genome-wide microarray analysis. With this approach, 3986 genes that are differently expressed between glomeruli from Pod1 KO and wild-type mice were identified, including a four-fold reduction of 8 integrin mRNA in glomeruli from Pod1 KO mice that was confirmed by immunostaining. This procedure may be adapted to any transgenic strain, providing a rapid and efficient method to dissect the function of specific genes in glomerular development.

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