Effective Silencing of Sry Gene with RNA Interference in Developing Mouse Embryos Resulted in Feminization of XY Gonad
Figure 3
Factors affecting the shRNA deliver efficiency and toxicity on developing embryos in vivo. shRNA vector transfection rate was measured by GFP expression level, and the toxicity was assessed by pregnant mice abortion rate and dead embryo rate. GFP expression level and dead embryo rate were detected after treatment with plasmids for 48 h, and pregnant mice abortion rate was detected at 48 h after injection. (a) Amount of PEI affects pDNA transfection rate and toxicity. 0.8 mg of pSilencer4.1/Sry565 plasmid mixed with same amount of (PEI+) or without PEI (PEI−) diluted in 1.0 mL 5% glucose solution was injected into 9.5 dpc pregnant mice tail vine in 10 seconds. (b) Effect of pDNA amount on transfection rate and toxicity. Certain amount of pSilencer4.1/Sry565 plasmids mixed with PEI, diluted in 1.0 mL of 5% glucose solution, were injected into 9.5 dpc pregnant mice tail vine in 10 seconds. (c) Effect of injection speed on transfection rate and toxicity. Pregnant mice at 9.5 dpc were intravenously injected with 0.8 mg of plasmid DNA and PEI complex in 1.0 mL saline. The injection time was varied from 5 to 30 s. (d) Effect of injection volume on transfection rate and toxicity. 0.8 mg of plasmid DNA and PEI complex was diluted in 0.2 to 2.0 mL 5% glucose solution and injected into pregnant mice at 9.5 dpc. (e) Effects of injection time on transfection rate and toxicity. 0.8 mg of plasmid DNA and PEI complex was diluted 1.0 mL 5% glucose solution and injected into pregnant mice various at 5.5 dpc to 10.5 dpc. GFP expressions in embryos and dead embryo rate were detected at 11.5 dpc. Pregnant mice abortion rate was measured in 48 h after injection.
