55 CONSTRUCTION AND IN VITRO DEVELOPMENT OF FOLLISTATIN TRANSGENIC PORCINE EMBRYO BY SOMATIC CELL NUCLEAR TRANSFER

Abstract

Data from mice showed that follistatin can block myostatin activity in vivo and follistatin overexpressed in transgenic mice can induce dramatic increases in muscle mass. In order to determine whether this effect of follistatin have the same results in swine, we design experiment to make a follistatin transgenic porcine by somatic cell nuclear transfer. We hope to get a follistatin transgenic porcine that has double muscle mass. The present study aimed to construct follistatin transgenic embryos by somatic cell nuclear transfer. A follistatin (FST) expression vector, pFST-1, which contains a porcine FST cDNA (with modification of codon usage) driven by the human cytomegalovirus (CMV) promoter, was constructed. A SV40-neo expression cassette as a selection marker was inserted into pFST-1 to generate pST101, which was transfected into early-passage male primary porcine fetal fibroblast cells by the lipofection method; the transfected cells were selected with 400 mg mL–1 G418. The G418-resistant colonies were pooled. Cell genome DNA was extracted and PCR analysis showed that CMV promoter, follistatin CDS, and SV40 polyA can be detected, indicating that the expression construct had integrated in cell genome. RT-PCR analysis showed that pST101 cells contained higher amounts of FST cDNA compared with the non-transfected cells, indicating that the follistatin was overexpressed in the primary porcine cells. Then, pST101 cell as donor cell and non-transfected cells as control donor cell were used to construct somatic cell nuclear transfer (SCNT) embryos. All of these SCNT embryos were cultured in Porcine Zygote Medium-3. The cleavage rate and blastocyst rate was assessed on 48 h and Day 6. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC, USA). The cleavage rate of pST101 cell and control cell derived SCNT embryos were 83.6 ± 2.0% (267/318) and 84.0 ± 2.2% (139/167) respectively, there were no significant (P > 0.05) differences between them. The blastocyst rate from pST101 cell and control cell derived SCNT embryos were 21.4 ± 1.8% (65/318) and 15.0 ± 3.0% (26/167) respectively, the SCNT embryos derived from pST101 cell showed a higher (P < 0.05) blastocyst rate. These results indicated that follistatin transgenic porcine embryo can be successfully constructed and can develop in vitro to blastocyst with a higher rate than control SCNT embryo. Further study will be focused on the full development in vivo of the transgenic embryos and phenotype of transgenic piglets. This work was supported in part by the National High-tech Research and Development Program of China (2008AA101006) and Major Projects for Transgenic Animals of China (2008ZX08006-002).