Production Of Cloned Transgenic Meishan Pigs By Using Somatic Cell Nuclear Transfer

Due to pig is more closer to humans rather than mice in anatomy,physiology and genetics,as an important economic animal,pig is considered as the ideal man’s organ xenotransplantation provider and a major animal model for human disease research.And acquisition of pig organ and preparation of the model depend on embryo engineering to provide abundant material.Currently,the pig embryo engineering is not consummate,which is represented by somatic cell cloning,transgene, pluripotent stem cell technology.Especially for the starting materials,the quality of porcine oocytes and embryos obtained in vitro is not high,and which reflects porcine oocyte and embryo in vitro culture system is not ideal as expected.Therefore the culture system of pig oocyte and embryo in vitro need to further optimize.This research firstly attempt to add recombinant human granulocyte-macrophage clony stimulating factor （hGM-CSF）,basic fibroblast growth factor （bFGF）,to obtain efficient,economical and stable porcine oocyte/embryo formulation in vitro culture.Later,with the practical need of long-distance transport for embryo,a more efficient,economical and practical embryo transportation equipment is designed,and it can be laied the foundation of efficiency of embryo production and production of transgenic cloned pig.Experiment Ⅰ.The study was to make a rapid sex-identification of porcine fetal fibroblast cells by double-gene PCR method, by designing primers in sex chromosome specific fragments.Seven Meishan fibroblast cell lines were extracted genomic DNA.According to X,Y chromosome specific fragment Sry and ZFx/y gene,two specific primers were designed for rapid fibroblast cell lines sex-identification with PCR method.Msz4,msz5,msz11,msz15,four of male cell lines and msz6,msz9,msz14,three of female cell lines,were identified successfully.Experiment Ⅱ.At first we investigated the effect on porcine oocyte maturation when hGM-CSF were added into the non-definitive maturation medium （PIVM）.The experimental group was added2ng/ml,10ng/ml,50ng/ml,100ng/ml of hGM-CSF,and the control group didn’t add hGM-CSF（0ng/ml）.We found there were no significant effects on the oocyte maturation rate when PIVM added2ng/ml,10ng/ml,50ng/ml hGM-CSF.But parthenogenetic blastocyst rate from matured oocytes of100ng/ml hGM-CSF group was significantly decreased compared with the control group （34.83±5.50%vs.58.83±4.55%， P<0.05）,there was no significant change between the two group on oocyte maturation rate.Then,to explore the true impact on porcine oocyte maturation,we added 2ng/ml,10ng/ml,50ng/ml of hGM-CSF （0ng/ml as the control group）into the chemical definitive medium （PIVM without any protein ingredients）.There was no significant difference between the groups（53.63±4.68%，45.38±3.03%,49.63±1.35%vs52.13±2.72%, P>0.05）.And there was also no significant difference between groups parthenogenetic blastocyst later development from matured oocytes（25.88±3.69%,29.87±4.10%,24.63±5.27%,23.75±3.63%，P>0.05）.We further examined the effect of different concentrations hGM-CSF on porcine parthenogenetic embryos in vitro culture （2ng/ml,10ng/ml,50ng/ml of hGM-CSF were added into PZM3and the control group was without hGM-CSF）.The result showed that there were no significant acceleration on cleavage rate,blastocyst rate,hatched blastocysts rate during embryo culture when hGM-CSF added into non-definitived embryo culture medium PZM3（P>0.05）,but adding10ng/ml,50ng/ml of hGM-CSF group can increase the blastocyst total cell number（79±7vs79±8vs48±5，P＜0.05）.Embryo cleavage rate（90.43±2.41%,90.43±2.41%,90.57±2.82%,90.57±1.90%，P>0.05）,blastocyst rate （18.57±5.48%,16.14±4.70%,18.57±4.47%,17.86±5.61%，P>0.05）were not improved when hGM-CSF added to chemical definitive embryo culture medium PZM4.Experiment Ⅲ.The aim of this experiment was to study the impact of bFGF on oocyte maturation,in order to filter out the proper concentration and addition time of bFGF which beneficial to improving the quality of porcine oocyte maturation.The experimental group was added2ng/ml,10ng/ml,50ng/ml of bFGF while bFGF-free culture medium was control group.The results showed that,10ng/ml of bFGF can significantly increase the maturation rate of oocytes （84.17±3.51%vs68.83±3.72%，P<0.05）,parthenogenetic blastocyst rate（56.67±3.96%vs34.33±4.57%，P<0.05）, hatched blastocyst rate （10.50±4.99%vs0.00±0.00%，P<0.05）and blastocyst total cell number（65±4vs34±4， P<0.05）.In order to investgate the effect of bFGF on porcine parthenogenetic embryo post development,2ng/ml,10ng/ml,50ng/ml of bFGF was added into embryo culture medium（control group without bFGF）.Oocytes conventional in vitro maturation were parthenogenetically activated and then cultured in experimental groups. Embryo cleavage rate,blastocyst rate, hatched blastocyst rate,blastocyst total cell number were consolidated to determine the role of bFGF.The results showed that there had no obvious effect on parthenogenetic embryos post-development when bFGF added into non-chemical definitive medium PZM3.There was a rising trend between parthenogenetic blastocyst rate （0ng/ml,2ng/ml,10ng/ml,50ng/ml,25.40±7.90%,37.60±6.52%,36.40±3.52% ,29.40±3.43%，P>0.05）and hatched blastocyst rate（0ng/ml,2ng/ml,10ng/ml,50ng/ml，0.00±0.00%,0.00±0.00%,1.60±1.60%,0.00±0.00%,P>0.05）,although it didn’t have significant difference.Experiment Ⅳ: We tried to develop a simple transport device to replace the expensive commercial embryo transporter. When Continuous culture laboratory CO₂incubator as control group, the effects of transit time on embryo development were revealed using self-made embryo transporter. The results show that, in addition to self-made embryo transporter2h groups, the blastocyst rate of other groups are slightly higher than the control group （P>0.05）. Self-made embryo transport device groups of different transport time had hatched blastocysts, especially2h group than other groups （P <0.05）. The results of blastocyst differential staining showed that ratio of ICM/total cells in2h-transport group,3h-transport group from self-made simple transport device and4h-transport group from conventional cell culture transporter were significantly higher than other groups（P<0.05）,including the control group.Experiment Ⅴ: In the second half year of2010，Meishan transgenic cell line （msz6-transfected pEGFP-N1gene） as the donor cells, a total of1080constructed embryos were transplanted to nine Wanbei black pig’s oviduct,And one pig was pregnant then termed delivery with the birth of four healthy piglets. After testing by PCR method, all of them were transgenic cloned pigs.In summary,at first this study identified the gender of the seven Meishan fibroblast cell lines by SRY-PCR method rapidly and accurately.Then we found that adding hGM-CSF didn’t significantly promote oocyte maturation and embryo development, and adding10ng/ml of bFGF significantly increased the oocyte nuclear maturation and cytoplasmic maturation, but there had no significant effect on embryo development and quality when bFGF only added to embryo culture medium.Self-made simple embryo transport device was economic and easy to operate, and the effectiveness would be perfect when transporting embryo in3h. Under established platform, we obtained the first cloned transgenic Meishan pigs by somatic cell nuclear transfer.