Rapid isolation of glomeruli coupled with gene expression profiling identifies downstream targets in Pod1 knockout mice.
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 alpha 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.
Cui
Shiying
S
The Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada.
Li
Chengjin
C
Ema
Masatsugu
M
Weinstein
Jordan
J
Quaggin
Susan E
SE
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007668:Kidney / Q000002:abnormalities; D007678:Kidney Glomerulus / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D018345:Mice, Knockout; D050199:Podocytes / Q000166:cytology / Q000502:physiology; D015854:Up-Regulation
D051792:Basic Helix-Loop-Helix Proteins; C112923:Tcf21 protein, mouse