Abstract：Pig induced pluripotene stem cells(iPSCs) are ideal materials for regenerative medicine and cell biology research because of their self-renewal and multilineage differentiation potential. In order to explore the embryo chimeric ability of pig iPSCs, the piggyBac transposon vectors, PB[Act-RFP] DS and Act-PBase, were used to cotransfect pig iPSCs. The PS24-R, a pig iPS cell line, was successfully obtained which expressed red fluorescent protein (RFP). We have got chimeric embryos through microinjecting the PS24-R cells into the 4~8 cell parthenogenetic embryos in vitro. Then we collected the statistics of blastocyst rate and chimeric rate as the chimeric embryos developed to the blastocyst stage. The result showed that the PS24-R cell using piggyBac vectors to transfect the iPS cells could stably express RFP. The pig iPS cells could be effectively observed in chimeric embryos. We also tried to microinject one, five, ten PS24-R cells and one PS24-R clone respectively to pig embryos to produce chimeric embryos. Along with increasing of the number of donor cells, the chimeric rate of embryos gradually increased, whereas, blastocyst rate declined. Compared with the parthenogenetic embryos, the expression lever of the stem cell pluripotent factors, OCT4(octamer binding transcription factor 4), SOX2(sry related HMG box-2) and NANOG, had significant upregulated in chimeric embryos. In summary, the pig iPS marked by PiggyBac vector can achieve chimerism in the pig early embryonic development stage. This work lays a foundation for the production of pig iPS chimeric animals.
Bradley A., Evans MJ., Kaufman MH., et al., 1984. Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines [J]. Nature, 309(5965): 255~256Campbell, K. H., P. Loi, et al., 1996. Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev. Reprod., 1(1): 40~46Chen W S, Han R, Shang K G, 1999. Formation of germline chimeras from murine embryonic stem cells. Acta Genetica Sinica, 26(2): 126~134（陈伟胜, 韩嵘, 尚克刚，1999. 小鼠ES 细胞种系嵌合体的获得. 遗传学报, 26( 2): 126~ 134）Cheng, D., Z. Li, et al., 2012. Kinetic Analysis Of Porcine Fibroblast Reprogramming Towards Pluripotency By Defined Factors. Cell ReprogramDing S., Wu X. H., et al., 2005. Efficient transposition of the piggyBac resource (PB) transposon in mammalian cells and mice. Cell, 122(3): 473~483Esteban, M. A., J. Y. Xu, et al. (2009). "Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig." Journal of Biological Chemistry 284(26): 17634~17640Evans M., Kaufman M, 1981. Establishment in culture of pluripotential cells from mouse embryos[J]. Nature, 292: 154~156Ezashi, T., B. P. Telugu, et al., 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci, USA, 106(27): 10993~10998Ezashi, T., B. P. V. L. Telugu, et al.. 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the United States of America, 106(27): 10993~10998Gardner RL., 1968. Mouse chimeras obtained by the injection of cell into the blastocyst[J]. Nature, 220(167): 5596~597Martin G. R., 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Proc. Natl. Acad. Sci. USA, 78: 7634~7638Mintz, B., 1967. Gene control of mammalian pigmentary differentiation. I. Clonal origin of melanocytes. Proc Natl Acad Sci,USA, 58(1): 344~351Montserrat, N., E. G. Bahima, et al., 2011. Generation of pig iPS cells: a model for cell therapy. J Cardiovasc Transl Res, 4(2): 121~130Mueller S., Prellek, Rieger N., et al, 1999. Chimeraic pigs following blastocyst injection of transgenic porcine primordial germ cells[J]. Mol. Repord. Dev., 54: 244~254Shim H., et al. 1997. Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Biology of Reproduction, 57: 1089~1095Saburi S., Azuma S., Sato E., Toyoda Y., Tachi C., 1997. Developmental fate of single embryonic stem cells microinjected into 8-cell-stage mouse embryos. Differentiation, 62: 1~11Takahashi, K. and S. Yamanaka, 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4): 663~676Wu B Y, Xian M W, Shang K G, Wu H L, 1995. The analysis of GPI in chimeric mice of ES cells(MESPU 13). Acta Genetica Sinica, 22(5): 336~342（吴白燕,冼美薇,尚克刚,吴鹤龄，1995. ES 细胞(MESPU13)嵌合体小鼠的GPI分析. 遗传学报, 22(5): 336~ 342）West, F. D., S. L. Terlouw, et al., 2010. Porcine Induced Pluripotent Stem Cells Produce Chimeric Offspring. Stem Cells and Development, 19(8): 1211~1220West, F. D., E. W. Uhl, et al., 2011. Brief report: chimeric pigs produced from induced pluripotent stem cells demonstrate germline transmission and no evidence of tumor formation in young pigs. Stem Cells, 29(10): 1640~1643Woltjen, K., I. P. Michael, et al., 2009. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 458(7239): 766~U106Wood, S. A., N. D. Allen, et al., 1993. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365(6441): 87~89Wu, Z., J. Chen, et al., 2009. Generation of pig induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol, 1(1): 46~54Yin, H. Q., H. G. Cao, et al., 2010. Generation of Induced Pluripotent Stem Cells From Porcine Fibroblasts. Progress in Biochemistry and Biophysics, 37(6): 607~612
