Formation Of Porcine Tetraploid Embryos And4N-2N Chimeric Embryos

Chimera is chimeric organism structure composed of different genetic cells or tissues. In mouse chimerism thattetraploid embryos produced by electrofusion mosaic with diploid embryos or ES, cells derived of tetraploid embryoscontribute to the extraembryonic lineages, cells derived of diploid embryo can contribute to embryonic andextraembryonic lineages, after transplant into receipt, the born pups can be completely from the diploid embryos or ES,this method is known as tetraploid compensation technique. Therefore, the tetraploid compensation technique canbypass the traditional diploid chimeric mice, obtain genetically modified mice directly. In addition, chimerism studieshelp to study embryonic cell development and differentiation and special species gene preservation, besideschimerism studies also have widespread application value in animal breeding, production of transgenic animalresearch, medical, industrial application fields.However chimera studies on porcine are rarely reported, how to optimize the production method of pig tetraploidembryos, when is best period for produce2N/4N embryo chimeric, as well as how cells of different ploidy distributein chimeric blastocysts is still not clear.Therefore, in order to optimize the methods of produce porcine tetraploid andtetraploid chimera;observation the biological phenomena of tetraploid mosaic, lay the theory foundation of establishpig disease model and transgenic animal production methods, we carried out the research of pig tetraploid andtetraploid mosaic.1. Optimization of fabrication of tetraploid embryo systemThe optimal electrofusion voltages for pig embryos at the two-cell stage is90volts, DC, the fusion rate (82.33±6.13), blastocyst rate (28.38±2.41), the mortality of the embryos (13.24±4.43).2. Process and quality detection of porcine blastocysts tetraploid embryo developmentPreimplantation development of embryo after electrofusion, the tetraploid later than diploid embryos beforemorula, but the compact and blastocyst formation time is the same. And there’s no significant difference inblastocyst rate between tetraploid and diploid,yet there is a significant difference of total cell number of4Nblastocyst between2N(45.34±5) and4N (21.90±4.95)3. Fluorescence in situ hybridization (FISH) to detect the ploidy of tetraploidPloidy of the embryos was detected by fluorescence in situ hybridization, detection fusion for tetraploid embryosdeveloped into blastocysts. There is68.18%tetraploid,27.27%diploid,4.5%for the2N/4N chimeric embryos.The control group of diploid blastocysts is in95%diploid,5%for the2N/4N chimeric embryos.4. The optimal period of aggregation of2N and4N The optimal period for2N/4N aggregation is4-Cell (4N)-4-Cell (2N) for blastocyst rate (74.36%), and cellnumber of blastocyst (67.10±15.39) was significantly higher than that of other experimental group.5. The distribution characteristics of different ploidy cells in2N-4N chimeric embryosWe aggregate204-Cell (4N)-4-Cell (2N),12developed to blastocysts, in10blastocysts the embryonic cellsderived of2N were mainly distributed in the ICM position, a small amount of the distribution to the location of TE;and in2blastocysts no any GFP signal, it should be not chimeric successful. In control,5of10develop to blastocyst,4N embryonic cells are in most of the TE site and outer of ICM.6. Chimerism of tetraploid embryo and IPS cellIn aggregation of “Sandwich”, IPS cells aggregate with two4N embryos successfully, the blastocyst rate was62.5%, the IPS chimeric blastocyst rate was45%. In microinjection, injection of IPS cells successfully chimeric to2N and4N blastocysts, chimeric rate of ips with2Nand4N embry was21.54%and29.79%respectively.In conclusion, we established the optimal sysem of produce pig tetraploid embryo and explored the chimeric oftetraploid with diploid embryo and IPS cells, which provides a foundation for further study of pig tetraploidembryos and tetraploid embryo chimera.