Effective Silencing of Sry Gene with RNA Interference in Developing Mouse Embryos Resulted in Feminization of XY Gonad
Figure 1
Schematic diagram of designing shRNAs expression vectors, and expression of siRNAs in embryos of pregnant mice. (a) Construction of shRNA expression plasmids. pSilencer4.1-CMV/neo vector was used as basic plasmid to construct the siRNA expression, plasmids pSilencer4.1/Sry565. The EGFP coding region was obtained from the pEGFP-N3 vector and cloned downstream of the SV40 promoter to replace the Neomycin gene to construct the plasmid. Gene-specific shRNAs (Sry565) or scrambled shRNA (not shown) were then ligated downstream of the CMV promoter. (b) Tail vein injection. The shRNA expression plasmids were delivered into pregnant mice though tail vein injection. (c) 11.5 dpc embryos were dissected from the uterus, and deciduas and membranes were removed. Transmitted light and fluorescence images of embryos were recorded using a Nikon-inverted fluorescence microscope to determine the extent of GFP expression. The left embryo was injected with pSilencer4.1/Sry565. The right embryo was injected with 5% glucose solution. (d) Analysis of EGFP expression in 11.5 dpc embryos. Semiquantitative RT-PCR was used to detect the GFP expression in different tissues, including liver, muscle, gonad, and limbs, in the absence (−) or presence of (+) pSilencer4.1/Sry565.
