Combinational electroporation and transplantation approach to studying gene functions in avian embryos

Gene transfection is an indispensable approach for studying gene function since it provides important information on gain-and/or loss-of-function.Chick embryos are also extensively employed for studying biological function since they are easily accessible and can be maintained alive after manipulation.The combination of both techniques presents a powerful approach to understanding how genes regulate embryo development.Furthermore,combining these approaches with tissue transplant techniques make even more attractive for elucidate gene function.Electroporation,employing parallelly fashioned electrodes,has been widely used in chick embryos.However,experimenters have been frustrated by unsuccessfully transfection in some embryonic tissue of interest because the electrodes were improperly positioned.We presently demonstrated the different patterns of organizing and positioning the electrodes,in combination with tissue transplantation,to efficiently and specifically transfect the chick embryonic head,trunk neural tube,heart tube,somites and neural crest cells with the GFP reporter gene.

基于鸡胚电转技术研究脊髓神经干细胞相关基因功能方法的建立

目的建立一种基于鸡胚电转技术研究脊髓神经干细胞（NSCs）相关基因功能的方法。方法 RT-PCR检测鸡胚发育不同时期脊髓NSCs表面标志物;在鸡胚胚龄（E）E2.5~E3时,利用活体电转基因技术将p CAGGSGFP质粒转染到鸡胚脊髓,E6时体视荧光显微镜下筛选绿色荧光蛋白（GFP）阳性胚胎,每组至少取材5个;通过脊髓横切及open-book技术观察神经纤维投射情况;普通光学显微镜下剥离出3~5条脊髓,经胰蛋白酶消化、离心后,无血清NSCs培养基重悬获得细胞铺板,于37℃、5%CO_2细胞培养箱内培养,定时观察GFP阳性脊髓NSCs的形态变化。结果 RT-PCR结果表明,鸡胚脊髓中阳性表达NSCs表面标志物;随后的脊髓横切及open-book结果表明,GFP阳性转染侧的神经纤维能穿过底板,投射到脊髓对侧;而脊髓NSCs体外培养结果显示,GFP标记的脊髓细胞具有典型的NSCs形态,继续培养后有明显突起产生。结论本实验成功建立了一种基于鸡胚电转技术研究脊髓神经干细胞相关基因功能的方法。