逆转录病毒法制备转基因鸡的方法分析

Method Analysis of Producing Transgenic Chicken Mediated by Retrovirus

随着生物制药业的迅速发展,转基因鸡已逐渐成为生物领域研究的热点之一。为建立和优化逆转录病毒法制备转基因鸡技术平台,以pHIT-Myc3为载体,以增强型绿色荧光蛋白(EGFP)为标记基因,采用共转染法包装泛嗜性VSV-G假逆转录病毒,浓缩后分别体外侵染鸡胚胎成肌细胞和体内胚盘下腔接种新生种蛋并孵化出雏。分别抽提成肌细胞、孵化5d的全胚和新生雏鸡不同脏器的基因组DNA进行PCR鉴定分析,结果分别在细胞和5d全胚胎及新生雏鸡的胸腺和皮肤中检测到了标记基因EGFP的存在,表明逆转录病毒法成功制备出转基因鸡,同时转基因鸡技术平台中的方法技术条件也得到了有效优化,为转基因鸡的进一步研究提供参考。

With the fast development of Biopharmacy,transgenic chicken is growing to be one hot spot of science study in the biology field. In order to build and optimize the technology and conditions of transgenic chicken platform produced by retrovirus, pHIT-Myc3 and enhanced green fluorescent protein （EGFP）gene were selected as vector and marker gene respectively in this experiment.Vesicular stomatitis virus G glycoprotein （VSV-G） pseudotyped retrovirus was packaged by co-transfecting method.Chicken fetal myoblasts（CFM） were infected and new laying zygotes were injected with concentration retrovirus and hatched. Genomic DNA of CFM,5d chicken embryos and different tissues of new-born chicken were extracted for PCR identification. Marker gene GFP was detected from CFM, 5d chicken embryos and thymus gland and skin of new-born chicken,which indicated transgenic chicken had been produced by retrovirus successfully.At the same time, the method and conditions of producing transgenic chicken had been optimized effectively,which provides references for further investigating transgenic chicken in the future.