Effective Silencing of Sry Gene with RNA Interference in Developing Mouse Embryos Resulted in Feminization of XY Gonad
Quantitative RT-PCR and Western-blotting analysis to detect the expression of gonadal GFP and Sry expression in embryonic mouse gonads. Gonads at 11.5 dpc were grouped on the basis of overall GFP expression observed at the macroscopic level. Quantitative RT-PCR was then carried out on grouped gonads. Gene expression was normalized to IL3 (autosomal in mouse) (mean ± SEM; ). (a), Relative expression of GFP in embryos. Relative high level of GFP expression was found in controls, and variable expression levels of GFP in male and female gonads with Sry knockdown. (b) Sry expression in control and Sry knockeddown gonads. (c) Western-blotting analysis of Sry expression. Control males which treated with 5% glucose solution or negative control vector (pSilencer4.1/EGFP), males embryos treated with Sry shRNA (pSilencer4.1/Sry565) and with low, medium, or high GFP expression, and the control females treated with control vector and show high GFP expression were all collected at 11.5 dpc and each gonad were separated for protein isolation. Western-blotting were performed to determine the SRY expression level in each group of embryos and β-actin was also be detected as a loading control. (d) Quantitative analysis of the Sry protein level. The expression of Sry was determined by Western blot. The value of Sry protein expression level was normalized to the β-actin expression level and the relative expression level of Sry in embryos which treated by 5% glucose solution was set at 100%. Results are expressed as the mean ± SEM, ().
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