Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.
RUAN WeiMinHAN JianYongLI PinCAO SuYingAN YangLIM BingLI Ning
目的使用RNA-Seq技术分析Miif-m敲除鼠与野生鼠的血管纹转录组，研究Mitf-m基因敲除后的差异表达基因及相关改变的分子机制。方法分别取Miif-m敲除鼠（Mitfmi-△M/mi-△M；MM组）与野生鼠（Miif-m＋/＋；ww组）的血管纹进行总RNA提取；制备cDNA文库，用Illumina HiSeq 2000测序系统行高通量测序。利用软件Tophat2．2．0将cleanreads比对到参考基因组；分别用软件RSeQS-2．3．2和cufflinks来检测测序结果的均一性和基因表达水平。使用edgeR和DESeq程序筛选差异表达基因（differential expressed genes，DEGs）；选择下调DEGs，用KOBAS2．O进行基因本体（geneontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）代谢途径的富集分析。结果MM组与WW组在参考基因组上mapping的cleanreads数分别为42463888和39718542，分别有88．7％和87．4％的reads是唯一映射，说明RNA-Seq结果可靠。DEGs筛查结果表明，相对于WW组，Mftf-m敲除鼠血管纹有45个DEGs，上调表达基因有7个，下调表达基因有38个。GO富集分析发现下调表达基因与黑色素的合成和代谢过程、色素沉着有关，值得关注的是与内向整流钾离子通道Kcnj10和Kcnj13相关。KEGG富集分析显示下调表达基因主要富集于黑色素生成通路。结论RNA-Seq分析拓展了Mftf-m基因调控网，为探究Mitf-m突变所致听力-色素综合征的分子机制及特异性研究Mitf不同转录子的功能奠定了基础。
