The Isolation And Culture Of Fetal Fibroblast Of Porcine And MSNT Gene Knockout

Gene targeting is a kind of new technology that developed after 1980'based on homologous recombination. Using this technology we can alterspecific endogenous genes that mean we can change the genetic information ofthe animals at will. Through specific mutating the gene of the animals, we canknow more about the function of the gene, set up animal models of humandisease, improve animal stain and built up bioreactors. At the early of 1980s',the successful isolation and culture of embryonic stem (ES) cells make itpossible for us to knockout a specific gene.At 1985, the phenomenon of homologous recombination was firsttestified in mammalian, it is the theory basic of gene targeting. From that day,transgenic cloning technology developed rapidly, kinds of animals were cloned,including mice, sheep, cow and pig. The technology is becoming morepracticed .However, the isolation of ES cells of big mammalians is difficult,and so somatic cell cloning became more useful. Somatic cell cloning is atechnique in which the nucleus of a somatic cell is transferred into anenucleated oocyte for the generation of a new individual, genetically identicalto the somatic cell donor. This technique gives us a new way to generatecloning big mammalians. Now, the genome of human beings has been sequenced, we have walkedinto the times of Functional Genomics. Gene targeting is becoming one of themost direct and effective method to study the function of genes. Furthermore,we can choose different gene targeting strategy for different purpose. Such as,complete knockout strategy, gene trapping strategy, hit and run strategy,conditional knockout strategy, etc.Myostatin is one member of transforming growth factors beta(TGF-β) family which is also called growth and differentiation factor–8(GDF-8) family. Myostatin genes encode secreted factors that are importantfor regulating embryonic development and keeping homeostasis in adult tissue.It plays an essential role in regulating skeletal muscle growth. In the nature,the Belgian blue and Piedmontese have double muscle phenotype, it is becausethe mutation of Myostatin , so we can modify the gene without influence thedevelopment of the animals. And now, somebody has cloned mice whichMyostatin gene has been knockout, the muscle of the cloned mice is muchbigger than the normal ones. So, it is feasible for us to make Myostatin geneknockout pigs. In our experiment , It is because this kind of cells is easy to beisolated, easy to be cultured for long time in vitro and the karyotyping of thecells is very stabile, so we choose fetuses fibroblast as donor cells .Porcinefetuses were obtained via hysterectomy of a pregnant(37d) gilt;Removal oflimbs, heads and internal organs, fetuses were finely chopped into piecesmeasuring about 1mm2. Digested with Liberase Blendzyme 3 (Roche 1814176)at 39.5℃ for about 7h;cultured individually at 39.5 ℃ , 5% carbondioxide(CO2). The cells were transfected with LipofectamineTM2000, andselected with G418 for about 7 days, GANC for about 10 days. The positivecells were identified by PCR, southern hybridization;Sex identification andkaryotyping were also be done. After identification, the positive cells wereused for nuclear transfer. The donor cells were inserted into enucleated ooytes,artificial activation of the reconstructed ooytes, and the reconstructed ooytescan develop into blastula in vitro.