Constract Of Myostatin Knockout Porcine Fetal Fibroblast And Preliminary Study On Myostatin Knockout Of Porcine

Myostatin, a transforming growth factorβ(TGF-β) family member, is a potent negative regulator of skeletal muscle growth. Myostatin genes encode secreted factors that are important for regulating embryonic development and keeping homeostasis in adult tissue. It plays an essential role in regulating skeletal muscle growth. Blocking myostatin activity may have the development of novel muscle enhancing agents for both the therapy for the human diseases such as HIV infects and agricultural applications.Naturally occurring myostatin mutations in the Belgian blue and Piedmontese display a similar double-muscled phenotype, confirming a role for myostatin in muscle development and regulation. Knockout the myostatin gene in mice results in a significant increase in skeletal muscle mass and a reduction in body fat. So we can modify the gene without influence the development of the animals. It is feasible for us to make Myostatin gene knockout pigs.Gene targeting is the most powerful technology for genetic manipulation in mammals. Using this technology we can alter specific endogenous genes, that means we can change the genetic information of the animals at will. Through specific mutating the gene of the animals, we can know more about the function of the gene, set up animal models of human disease, improve animal stain and built up bioreactors. At the early of 1980s', the successful isolation and culture of embryonic stem (ES) cells make it possible for us to knockout a specific gene .At 1985, the phenomenon of homologous recombination was first testified 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 more practiced.However, the isolation of ES cells of big mammalians is difficult, so somatic cell cloning became more useful. Somatic cell cloning is a technique in which the nucleus of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. This technique gives us a new way to generate cloning big mammalians. Although somatic nuclear transfer cloning has helped us answer many fundamental questions in developmental biology, and has many potential applications, the efficiency of somatic cell nuclear transfer is very low. Scientists are currently working on improving the efficiency of the procedures, and applying the technology to the creation of animals with targeted genetic modifications.Now, the genome of human beings has been sequenced, we have walked into the times of Functional Genomics. Gene targeting is becoming one of the most 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.In our experiment, It is because this kind of cells is easy to be isolated, easy to be cultured for long time in vitro and the karyotyping of the cells is very stabile, so we choose fetuses fibroblast as donor cells. Porcine fetuses were obtained via hysterectomy of a pregnant (37d) gilt. Removal of limbs, heads, tails and internal organs. The fetal fibroblasts were cultured by fetal tissue pieces, which digested with collagenase. Establish an ideal cellular culture method. After several passage culture and freeze-thawed,the fetal fibroblast cells still have normal capacity of passage. Karyotype analyze indicate that after 20 passage of in vitro culture no abnormal. It suit for the need of clone and transgenic clone. We established the technique of sex determination of fetal fibroblast of porcine by PCR.G418 cell toxicity test of the cell line show that, when concentration of G418 was 250μg/ml, the normal cell died on 10 day. So, we adoption 250μg/ml G418 when selection of transfection. The cells were transfected with LipofectamineTM2000, and selected with G418 for about 7 days, G418 and GANC for about 10 days. The positive cells were identified by PCR, southern hybridization, Sex identification and karyotyping was also been done. After identification, the positive cells were used for nuclear transfer. In order to improve efficiency of PCR amplification, we design their pair of primer. On lateral of neo gene and short arm have one pair of specificity primer. On neo gene have one pair of primer and on TK gene have another. PCR amplification of positive cell are 2995bp,409bp and no amplification. The PCR identification result of resistance cell is same as before. So we conclude that the cell occurrences homologous recombination. Southern blotting analysis use DIG High Prime DNA Labeling and Detection Starter Kit II, The Kit efficiency of probe labeling and sensitivity of immunodetection are high. We design their probe. The experimental result showed that normal cell gene have 2727bp and resistance cell is have 3364bp. The length of resistance cell is same as positive cell. So, we have a conclusion: This cell line is the of MSTN gene knockout fetal fibroblast of Changbai porcine. Sexing show that this cell line is female.For pig nuclear transfer, matured oocytes are needed in large numbers and in vivo matured oocytes are very expensive to acquire. Thus many has chosen to use in vitro matured oocytes. By using this defined system a large number of oocytes can be synchronized in a specific development stage. So we chosen to use in vitro matured oocytes. Immature oocytes are derived from ovaries obtained from the slaughterhouse and subsequently matured in vitro. Matured oocytes eliminate first polar body after 44 hours. Maturing rate is 71.80%. We use in vitro matured oocytes. Direct injection of the donor cells into the cytoplasm of enucleated oocytes. After electrical activation,30mins later, The fusion percent is 75.70%. After electrical activation of the reconstructed ooytes and 7d culture in vitro, the reconstructed ooytes can develop into blastula in vitro, and the percent is 31.56%. The PCR identification result of blastula was same as the positive cell. So we have a further conclude: This blastula is the MSTN gene blastula of Changbai porcine. Transfer embryos into the oviduct of the 10 porcine by 18-22h cultures of reconstructed ooytes. After 37d, one was pregnant. But operation after 114d of the pregnant, the pregnant surrogates lost their fetuses. We predicated fetal abortion. The reason of fetal abortion needs further study.