New techniques are being applied to identify all the genes involved in mammalian gonad development and differentiation. As this list of genes increases, understanding the potential interactions between these genes will become increasingly difficult. We used a real time reverse transcription PCR (real time RTPCR) protocol to examine and compare the relative expression levels of 55 genes in individual mouse fetal gonads. Real time PCR analysis demonstrated that except for Sry, no differences in relative gene expression were detectable between XX and XY gonad/mesonephroi complexes at embryonic day (E)11.5. Following Sry peak expression at E11.5, a number of genes were expressed at significantly higher relative levels in E12-14 XY than XX gonads. Of six genes expressed at higher levels in E12.5-14 XX than XY gonads, three, Bmp2, Emx2, and Fgfr2, had not been reported previously. Our results caution that differential localization patterns observed with whole mount in situ hybridization techniques may not accurately reflect changes in transcript levels. We conclude that real time PCR is an efficient and powerful tool for studying multiple gene expression patterns during gonad development and differentiation, and can provide insight into gene interactions.
Bibliographical noteFunding Information:
The authors thank Shelly Wang for help with tissue processing, and Jeff Budzik and Trudy Radcliffe from the Microchemistry Department at The Jackson Laboratory for the RNA quality and quantification analyses. We are especially indebted to Derry Roopenian for help with the GPR analysis, and Daniel Shaffer for advice on primer design and sample preparation. In addition, we thank Mary Ann Handel, Timothy O'Brien, Derry Roopenian, and Daniel Shaffer for critically reading this manuscript and providing helpful suggestions. The Jackson Laboratory is AALAS accredited and all animal procedures were approved by The Jackson Laboratory Animal Care and Use Committee. This study was supported by NIH grants GM20919 (E.M.E) and HD07065-25 (G.J.B.), and NCI core grant CA34196 (The Jackson Laboratory).
- Gonad differentiation
- Multi-gene platform