CRISPR/Cas9介导的外源基因靶向插入鸡EAV-HP基因组

CRISPR/Cas9-mediated foreign gene targeted knock-in into the chicken EAV-HP genome

摘要:

本研究旨在通过CRISPR/Cas9介导外源基因靶向插入鸡EAV-HP基因组。首先设计特异性引物并扩增鸡内源性病毒 (EAV-HP) 左右同源臂和增强型绿色荧光蛋白 (eGFP) 基因表达盒，然后通过重叠延伸PCR技术将两个同源臂DNA连接至eGFP表达盒两侧，获得全长DNA片段LER，并克隆至pMD19-T载体，获得携带eGFP基因的供体载体pMDT-LER。随后在HEK293T细胞中验证供体载体pMDT-LER能成功表达eGFP后，将EAV-HP打靶载体和供体载体共转染至DF-1细胞，观察绿色荧光阳性细胞，提取细胞基因组，PCR检测外源基因eGFP成功整合至鸡基因组EAV-HP位点。最后，将转基因细胞DF-1传至第7代，用PCR和Western blotting检测eGFP在转基因细胞中稳定表达。文中初步验证外源基因eGFP能整合至鸡EAV-HP位点并稳定表达，为转基因鸡的研究提供新整合位点。

Abstract:

The study aims to use CRISPR/Cas9 introducing foreign gene targeted knock-in into chicken EAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein (eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9 expression vector and pMDT-LER into chicken DF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site for research of transgenic chicken.