转基因DT40细胞导入早期鸡胚后的分布及绿色荧光蛋白表达的研究

目的为了研究经过基因修饰的体细胞导入到禽类胚胎以后,供体细胞及外源基因是否能在受体胚胎中成活并且外源基因是否可以长期表达。方法筛选得到稳定整合绿色荧光蛋白基因的鸡DT40细胞作为外源蛋白的运载工具,通过血管微注射的方法将其导入到于38.5℃温度条件下孵化65～70 h的鸡胚中,并将操作后的鸡胚在原孵化条件下继续孵化。在孵化的不同时期取移植了DT40细胞的嵌合体胚胎在荧光显微镜下观察荧光细胞的存活与分布情况。并通过PCR以及免疫组织化学方法检测供体细胞在受体中的位置以及绿色荧光蛋白的表达情况。结果荧光标记的DT40细胞可以存活于受体不同的组织器官中,包括：脑、心脏、肝脏等。导入胚胎的整合外源基因的DT40细胞可以存活到胚胎出雏之前,并且外源基因能够正常表达。结论可以通过此方法将外源基因导入到受体中,并使目的蛋白在受体胚胎中持续表达,为胚胎期导入外源蛋白诱导免疫耐受的研究以及将转基因细胞移植到动物体内生产目的蛋白的研究提供科学依据和技术平台。

The Distribution and Green Fluorescent Protein Expression of Transfected Chicken DT40 Cells after Transplanted into Early Chicken Embryos

Objective To investigate whether the genetically modified somatic cells can survive and express foreign gene for a long time after being transplanted into avian embryo.Methods Chicken DT40 cells as a cell vehicle for delivering foreign protein were transfected with the green fluorescent protein（GFP） gene,and were introduced into early chicken embryos via blood vessel microinjection at 65~70 h of incubation at 38.5 ℃.The manipulated eggs were continued to incubate at same condition.The chimerisms of the transplanted DT40 cells were preliminarily observed under fluorescence microscopy at the different stages in the embryos and the hatchlings.Meanwhile,the chimeric positions of the donor DT40 cells and the expression of GFP were further examined by polymerase chain reaction（PCR） and immunohistochemistry.Results The results showed that fluorescent-labeled DT40 cells embed in the different organs of the recipients including brain,heart,liver,etc.and can survive before chicken hatch and the GFP gene can be expressed normally.Conclusion Long-term survival of the donor DT40 cells in recipient and normal expression of the GFP gene imply that this approach can be explored for continuous production of target protein in the host chicken,which may provide a basis for avian immune tolerance research and the production of bioreactor.