体外培养的鸡原始生殖细胞在早期胚胎迁移的研究-动物遗传育种与繁阳殖专业毕业论文.docx

数的增加，迁移的PGCs增加，当移植的PGCs多于10000个／胚时，迁 数的增加，迁移的PGCs增加，当移植的PGCs多于10000个／胚时，迁 移的PGCs不再增加。我们将移植10000个PGCs的鸡胚孵出，出壳4d 小鸡睾丸内发现外源GFP．PGCs，证明了外源GFP．PGCs能在受体性腺 内生长增殖。随后，成熟雄性嵌合体鸡精液DNA PCR结果显示，4个假 定嵌合体中有1个呈GFP阳性。最后，我们探究了CXCR4信号通路在 PGCs迁移过程中的作用。RT-PCR结果显示，PGCs表达了趋化因子受 体Cxcr4基因。此外，我们在移植的细胞滴中分别添加CXCR4信号通 路抑制剂AMD3100和WZ811，AMD3100浓度为0nM、10nM、50nM， WZ81 l为0nM、lnM、2nM，试验结果表明，50nMAMD3 100以及lnM、 2nMWZ811能明显抑制PGCs的迁移(PO．05)。证明了SDF．1／CxCIH 信号通路参与调控PGCs在早期鸡胚中的迁移和归巢。 综上所述，广西黄羽鸡PGCs能在体外长期培养，经转基因修饰后 仍保留其生物学特性。受体鸡胚孵育50h为最佳移植时间，每个鸡胚移 植10000个PGCs迁移至性腺的PGCs最多，归巢至性腺的PGCs能在 受体睾丸中正常增殖生长。SDF．1／CXCR4信号通路参与介导PGCs的体 内迁移过程。 关键词：原始生殖细胞转基因移植迁移 万方数据 MIGRATION MIGRATION OF IN VITRO．CULTURED CHICKEN PR．IMOR．D认L GERM CELLS IN EA R I Y EMBRYO AB STRACT As the precursors of gametes，primordial germ cells(PGCs)are responsible for delivering genetic information from one generation to the next． Chicken PGCs could be isolated from early embryo，expanded in vitro and genetically modified，then transplanted into recipient embryos to produce germline chimeras．Thus，PGCs is all ideal tool to generate transgene animal． However,production of transgene chimeras was in an inefficient process due to limited availability of knowledge and poor optimization of the migration and colonization ofPGCs to gonad．The objective ofthis study was to explore the factors which affect the efficiency ofthe migration ofchicken PGCs in the recipent embryos，and thus provide basis to optimize the procedure to produce transgenic chickens． Firstly,PGCs were isolated from circulating blood of Guangxi yellow feather chicken embryos at stage 1 3．1 5 and then cultured in vitro．After genetically modificatiol11catlon and screemnz。．，byby puromycin，ouromvcina stable PGCs line1 expressing green fluorescent protein (GFP) was established． Immunocytochemistry revealed that after in vitro culture and genetically modification，these PGCs were still positive for S SEA一1．RT-PCR analysis showed that PGCs express the Dazl，Cvh，Pour,Cdh and Nanog genes．These 万方数据 resu