title: A simple and efficient method to transfect small interference RNA into bovine SCNT embryos.
abstract: 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 bovine somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of platinum microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted DNA methyltransferase-1 (DNMT1) 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 DNMT1 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of DNMT1 messenger RNA, DNMT1 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.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: LiJun
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: WenZhe
authors.initials: W
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: QingFu
authors.initials: Q
authors.lastname: Mao
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: YongSheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Song
authors.initials: S
authors.lastname: Hua
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China. Electronic address: zhy1956@263.net.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002417:Cattle / Q000235:genetics*; D047108:Embryonic Development / Q000235:genetics; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D034622:RNA Interference; D034741:RNA, Small Interfering; D014162:Transfection / Q000379:methods / Q000662:veterinary*
chemical_list: D034741:RNA, Small Interfering
keywords: Bovine; Electrofusion; Preimplantation embryo; SCNT; Small interference RNA transfection method