The Study On The Cloned Goat Knockout FGF5Gene Transferred Somatic Cell

FGF5gene is a member of the FGF gene families, has an important regulating role in hair growth cycle, mutate FGF5gene can extend the growth period of the hair growth cycle, so as to increase the length of hair. This study intends to knock out FGF5gene in the Inner Mongolia white down producing goat and create goats that have high yield pashm using nuclear transfer. Firstly, we established the goat fetal skin fibroblast cell line that isolated and cultured by attachment of tissue pieces from Inner Mongolia white down producing goat fetal. Secondly, we designed three knockout vectors and were named as pLOX-EGFP-FGF5, RNAi-FGF5and TALEN-FGF5, respectively. Take these three knockout vectors to transfect the goat fetal fibroblasts cell use liposome mediated method, electroporation and Cell nuclear transfer apparatus respectively. Then we screening the positive cells and propagate them. Thirdly, we choose the positive cells as nuclear donor to building transgenic goats reconstructed embryos and transplanted into synchronous estrus recipients to making transgenic cloned goats.1. According to already announced cattle and goats FGF5gene sequence design primers and cloned Inner Mongolia white down-producing goat FGF5gene. The result shows that FGF5gene has three exons and two introns and the cDNA length of it is924bp. Then we design and build three kinds vectors, they were gene targeting vector pLOX-EGFP-FGF5, RNA interference vectors RNAi FGF5-1#and RNAi-FGF5-2*, and TALEN vectors TN000201, TN000202, TN000204respectively. Identification by sequencing and enzyme cut, the results showed that the vectors are correct and available.2. Using attachment of tissue pieces to established nine goat fetal skin fibroblast cell lines that isolated and cultured from Inner Mongolia white down-producing goat fetal. We determine the optimum concentration of G418screening in all cell lines by G418sensitivity experiments, and select the goat6and goat9as the screening transgenic positive cell lines because they have the highest G418resistance. Take these three kinds knockout vectors to transfect the goat fetal fibroblasts cell use liposome mediated method, electroporation and Cell nuclear transfer apparatus respectively, ultimately selected three kinds of genetically modified (gm) positive cell line.3. Use the three kinds genetically modified (gm) positive cell line as nuclear donor to build the transgenic cloned embryos respectively. Application of gene targeting technology we select embryos transferred into46recipients, and there were5pregnant goats and the pregnancy rate was8.7%. One lamb survived four days, two lambs died a few minutes after birth and only one lamb lived healthy. The expected date of confinement about the two recipients is in May. Application of RNA interference technology we select embryos transferred into143recipients, and there were24pregnant goats and the pregnancy rate was16.78%, but all goats’ pregnancy abortion. Application of TALEN technology we select embryos transferred into62recipients, by B ultrasonic examination found that5receptors were pregnant, and the pregnancy rate was8.06%.4. Application of gene targeting technology target except FGF5gene, use transgenic PCR appraisal the lambs, the result showed that the lamb in number083was double FGF5gene knock out and in good health. The other three lambs were non-gmo clone. In the application of RNA interference technology target except FGF5gene, by real-time fluorescent quantitative PCR to identification of aborted fetuses, the results show that the fetus FGF5gene expression was significantly lower. Application of TALEN technology target except FGF5gene transgenic cloned lambs, via PCR product sequencing appraisal, the results show that4target area of newborn lambs FGF5gene are missing two bases, causing FGF5gene frameshift mutations.