鸡TBX6过表达和干扰载体构建及干扰载体效率检测  

TBX6 Overexpression and Interference Vector Construction and Detection of Interference Vector Efficiency

作　　者：武嵘峰 陈欣雨 胡菜 耿晴晴 程富富 席一凡 左其生[1] 张亚妮[1] WU Rongfeng;CHEN Xinyu;HU Cai;GENG Qingqing;CHENG Fufu;XI Yifan;ZUO Qisheng;ZHANG Yani(Jiangsu Key Laboratory of Animal Genetics,Breeding and Molecular Design,College of Animal Science and Technology,Yangzhou University,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学动物科学与技术学院,江苏省动物遗传繁育与分子设计重点实验室,江苏扬州225009

出　　处：《中国家禽》2023年第10期12-18,共7页China Poultry

基　　金：江苏省研究生实践创新计划项目(SJCX22_1795);大学生科创基金项目(X20210648)。

摘　　要：研究旨在构建TBX6的过表达和干扰载体,为后续研究TXB6在精原干细胞(Sper⁃matogonial stem cells,SSCs)形成过程中的功能奠定基础。通过qRT-PCR检测TBX6在胚胎干细胞(Embryonic stem cells,ESCs)和SSCs的表达水平,根据NCBI上提供的TBX6 CDS区的序列,设计引物扩增目的片段,将其连接到经线性化的慢病毒载体pCDH-CMV-MCS-EF1-copG⁃FP-T2A-Puro上,构建TBX6的过表达载体;同时,根据TBX6 CDS区设计三个干扰靶点,在退火后将其连接到经线性化的干扰载体pLVX-shRNA2-puro上,并且进一步将三个干扰载体转染鸡成纤维细胞系(DF-1),观察荧光表达情况并通过qRT-PCR检测其干扰效率。结果显示,TBX6在SSCs上的表达量显著高于ESCs(P<0.05),测序和双酶切验证显示成功构建TBX6过表达及干扰载体;干扰载体转染DF-1细胞后均能稳定表达EGFP荧光蛋白,且shRNA2-TBX6的干扰效果最好。结果表明,TBX6过表达及干扰载体构建成功,且经定量检测后确定shRNA2-TBX6的干扰效果最好。This paper aimed to construct the overexpression and interference vector of TBX6,so as to lay an experimental foundation for the subsequent study of the function of TXB6 in the formation of spermatogonial stem cells(SSCs).Firstly,the ex⁃pression levels of TBX6 in embryonic stem cells(ESCs)and SSCs were detected by qRT-PCR.Secondly,according to the se⁃quence of TBX6 CDS region provided on NCBI,primers were designed to amplify the target fragment and connected to the linear⁃ized lentivirus vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro to construct the overexpression vector of TBX6.At the same time,three interference targets were designed according to TBX6 CDS region and connected to linearized interference vector pLVX-shRNA2-puro after annealing.The three interference vectors were further transfected into chicken fibroblast cell line DF-1 to observe the fluorescence expression and detect the interference efficiency by qRT-PCR.The results showed that the expres⁃sion level of TBX6 on SSCs was significantly higher than that of ESCs(P<0.05).Sequencing and double digestion verification showed that the overexpression and interference vectors of TBX6 were successfully constructed,and the interference vectors could stably express EGFP after transfection of DF-1 cells,and shRNA2-TBX6 had the best interference effect.The overexpres⁃sion and interference vector of TBX6 were successfully constructed,and shRNA2-TBX6 was determined to have the best interfer⁃ence effect after quantitative detection.

关 键 词：TBX6 过表达载体 干扰载体 

分 类 号：S831.2[农业科学—畜牧学]