Swines From Gene Knockout With Genes MSTN And GGTA1 By Homologous Recombination Vector And Somatic Cell Nuclear Transfer

To explore the production methods of gene knockout pig, this study was adopted a method combining somatic cell nuclear transfer technology with gene targeting to produce of MSTN gene knockout pig and GGTA1 gene knockout pig, thereby it can provide the basic data for pig transgenic breeding and human xenotransplantation studies. The results are as follows:1. Production of MSTN gene knockout pig. Homologous recombination with positive and negative targeting vector pLOXP-MSTN-KO was used to knockout the third exon of fetal fibroblast MSTN gene in large white pig. The targeting positive cell as donor cells to conduct nuclear transfer and the cloned embryos were cultured after fusion and activation. 1-cell and 2-4 cell stage MSTN^+/- cloned embryos were transferred into 10 and 5 recipient gilts, respecitively. After 35 days, 7(70%) and 2(40%) recipient gilts were pregnant by B-ultrasonic determination. After full-term, 27 and 4 piglets were borned. The Southern blot shown that all of the cloned piglets were MSTN^+/-. The efficiency of somatic cell clone was 1.0% and 0.4%, respecitively. The results showed that 1-cell was improved the pregnancy rate and the cloning efficiency. The season effected of the cloned piglets on survival. Summer is better than autumn (75% vs. 33%) in surrive.The fluorescence quantitative PCR detection of MSTN^+/- piglets in MSTN gene significantly decreased to 90%. Phenotype of MSTN^+/- pig hip muscle development significantly higher than control, others performance are being observed.2. Production of GGTA1 gene knockout pig. One allele of GGTA1 was knocked out by homologous recombination promoter deficient targeting vector pBS-GGTA1-SKO knockout in Wuzhishan inbred miniature fetal fibroblast. The targeting positive cells were used as donor cells for cloned embryos. 1-4 cell stage cloned embryos were transferred into 7 recipient gilts. After 35 days, 5(71%) recipient gilts were pregnant by B-ultrasonic determination and 20 piglets were borned. The Southern blot shown that all of the cloned piglets were GGTA1^+/-. The efficiency of somatic cell clone was 1.3%. One of the recipients in the present study produced 10 cloned piglets. The season effected of the cloned piglets on survival. Autumn birth is better than winter (75% vs. 33%) in surrive.3. The present study was designed to examine the effect of Scriptaid, a low toxicity deacetylase inhibitor, on developmental competence of cloned embryos and cloning efficiency. The cloned embryos were reconstructed with Wuzhishan inbred miniature pig fetal fibroblast cells as donor cells. We examined in virto the developmental competence of cloned embryos under various exposure times (0～36 hour) and concentrations (0～300nmol/ L).The result showed that the treatment with 100 nmol/L Scriptaid for 24 hour supported a higher blastocyst development rate than the control groups (30.4% vs.17.5%, P<0.05). In order to assess in vivo developmental competence, embryos treated with 100nmol/L Scriptaid for 24 hour and the control group embryos were transferred into 4 recipient gilts, respectively. Litter size and cloning efficiency of treatment group were significantly higher than those of control group (5 vs.1.5; 2.4% vs.0.7%; P<0.05). These date suggest that Wuzhishan inbred cloned embryos with 100nmol/L Scriptaid treatment for 24 hour can enhance their developmental competence and cloning efficiency.