基于TAIL-PCR分析外源基因在小鼠精原细胞中的整合位点  

Integration Sites of Exogenous Gene in Mouse Spermatogonia Based on TAIL-PCR

作　　者：冯美莹 曾梓凡 邝乃诵 温淑桦 刘文华[1] 王瑛华[1] FENG Meiying;ZENG Zifan;KUANG Naisong;WEN Shuhua;LIUWenhua;WANG Yinghua(School of Life Science,Zhaoqing University,Zhaoqing,Guangdong 526061,China)

机构地区：[1]肇庆学院生命科学学院,广东肇庆526061

出　　处：《肇庆学院学报》2022年第2期71-76,共6页Journal of Zhaoqing University

基　　金：肇庆学院青年项目(QN202123);肇庆学院实践教学改革研究项目(sjjx201910);广东省高等教育教学改革项目(粤教高函[2020]20号)。

摘　　要：外源基因在哺乳动物细胞的整合位点会影响宿主细胞的生殖发育,在基因功能研究中常常会先确定其整合的染色体位置.为研究外源基因在小鼠精原细胞中的整合位点,以转入Cas9基因的小鼠精原细胞为研究材料,通过TAIL-PCR克隆Cas9整合到基因组的旁侧序列,结合T载体连接和碱基测序技术在有限的基因组中检测Cas9整合到小鼠基因组的具体位置.结果表明,转入外源基因的小鼠精原细胞折光性好,生长良好,PCR扩增发现Cas9基因成功转入到小鼠精原细胞的基因组上;结合上游特异性引物和简并引物可扩增出2条特异性条带,测序发现均为载体序列;结合下游特异性引物和简并引物可扩增出4条特异性条带,其中有2条扩增片段属于载体序列,2条扩增片段为小鼠基因组12号染色体中的序列.这表明外源基因整合到小鼠精原细胞12号染色体上.The integration site of exogenous gene in mammalian cells will affect the reproductive development of the host cell.Normally,the chromosomal location of integration will be determined first in gene function research.To study the integration site of exogenous genes in mouse spermatogonia,we used mouse spermatogonia which transferred with Cas9 gene as the research material.The flanking sequence of Cas9 integrated into the genome were cloned by TAIL-PCR,combined with T Vector ligation and confirmed the sequence by base sequencing technologies,which could detect the specific location of Cas9 integration into the mouse genome.The results showed that the mouse spermatogonia which transferred with the exogenous gene had good refractive index and good growth.PCR amplification revealed that the Cas9 gene was successfully transferred to the mouse spermatogonia genome.Combining the forward specific primers and arbitrary degenerate primers can amplify 2 specific bands,which are all vector sequences found by sequencing.Combining the reverse specific primers and arbitrary degenerate primers can amplify 4 specific bands,of which 2 amplified fragments belong to the vector sequence,and 2 amplified fragments are the sequence of the mouse genome on chromosome 12.It is indicated that the exogenous gene is integrated into the chromosome 12 of mouse spermatogonia.

关 键 词：整合位点 TAIL-PCR 小鼠 Cas9 外源基因 

分 类 号：Q789[生物学—分子生物学]