A simple and efficient method to transfect small interference RNA into XXXX SCNT embryos.

@entity28319 @entity291 @entity902
RNA interference is an important tool to study the gene function. Microinjection and electroporation are usually used to transfer DNA, small interference RNA (siRNA), morpholinos, and protein into oocytes or embryos. This study used a simple and effective method to transfect siRNA into @entity291 somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of @entity902 microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted @entity28319 ( @entity28319 ) was chosen to verify the siRNA transfection efficiency of this approach. First, a suitable concentration of siRNA was mixed with Zimmermann's fusion medium. Reconstructed embryos were then added into the microdrops of the mixed fusion medium to simultaneously transfect the siRNA and electrofuse the SCNT embryos. Our results showed that transfecting @entity28319 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of @entity28319 messenger RNA, @entity28319 protein, and global DNA methylation levels in the SCNT embryos. Meanwhile, the survival rate after electrofusion (90.4% vs. 89.4% vs. 89.1%, P > 0.05) and developmental rates of the SCNT embryos (72.8% vs. 74.9% vs. 72.4%, P > 0.05; 29.7% vs. 31.7% vs. 29.7%, P > 0.05) were not significantly affected. In summary, siRNAs were effectively transfected into the SCNT embryos via the proposed method and exert their functions, and the normal development of preimplantation SCNT embryos was not affected by the method used.
