Production Of Transgenic Cloned Pigs With HOXA10by Somatic Cell Nuclear Transfer

Sows reproductive traits, especially the litter size, are the important factors that affect the economic efficiency in pig enterprises. Due to the litter size trait is the sex-limited character,or low heritability trait at the same time, through the traditional breeding method to improve litter size has been slow and ineffective. However, genetically modified(gm) technology is an effective way to solve the problem. Researches in other species of the cellular level and the mice individual level have showed that the Homeobox A10(HOXA10) is one of the most promising candidate genes.For the further research of HOXA10gene on individual level of animal reproductive performance,use somatic cell nuclear transfer produce the HOXA10transgenic pig was necessary. The results are as follows:1. Cells isolation using trypsin followed by digestion of the fetus tissue(Large White pig fetus at33d of gestation).7strains of Large White pig fetal fibroblast cell line were established.Among them,3strains fibroblasts were come from male fetus. Chosen one of the male fibroblasts to culture for drawing the growth curves. The results suggested that the cells of generation on F8, F10and F13were experienced the latent period, the logarithmic growth phase and plateau phase. So the cell line could be available as the donor cell of the transgenic cloning manipulation before the13th generation.2. The linearized pc3.1-HOXA10-DNA plasmid was transfected into the Large White pig fetal fibroblast cell by using Lipo2000mediated transfection method.After G418(800ug/ml) selection for7-8days, the cells in the negative control group were completely dead, while the experimental group with visible monoclonal cell circle formation. Expanded the monoclonal cells and established7strains of monoclonal cell line finally. Then extracted genome DNA of the monoclonal cell lines for positive cells detection. The result of PCR showed the7strains of monoclonal cell line were positive. Detected the Large White pig fetal fibroblast cell cycle and found that cells serum starvation3d and fully confluence on Gl phase were more than70%and the two groups have no significant difference on G1phase (P>0.05). 3. Selected4strains of monoclonal cells with good condition as nuclear donor cells and transplanted2-cell stage to4-cell stage embryos (>230) into estrus sows’ fallopian tubal ampullae.10sows were transplanted in all and3sows born8cloned pigs,6alived so far(8months of age).4. The result of PCR showed the8cloned pigs were positive. Absolute quantitative PCR was used to calculate the copy number of transgene and the results showed that the copy number were not less than3. And the genome walking result showed the No.2cell cloned pig located on chromo.14and No.3cell cloned pig located on chromo.4, chromo.11, chromo.15, chromo.18.