PGC介导的TMEM182基因敲除性腺嵌合体鸡的制备  

PGC-mediated Preparation of TMEM182 Knockout Gonadal Chimeric Chickens

作　　者：温华强 陈家辉 黄振文 钟菁 陈庚华 聂庆华[1,2] 张细权 张德祥[1,2] 陆阳清 罗文[1,2] WEN Huaqiang;CHEN Jiahui;HUANG Zhenwen;ZHONG Jing;CHEN Genghua;NIE Qinghua;ZHANG Xiquan;ZHANG Dexiang;LU Yangqing;LUO Wen(College of Animal Science,South China Agricultural University,Guangzhou,Guangdong 510642;Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding,Guangzhou,Guangdong 510642;College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004)

机构地区：[1]华南农业大学动物科学学院,广东广州510642 [2]广东省农业动物基因组学与分子育种重点实验室,广东广州510642 [3]广西大学动物科学技术学院,广西南宁530004

出　　处：《中国家禽》2024年第9期18-25,共8页China Poultry

基　　金：广东省重点领域研发计划项目(2022B0202100001);国家自然科学基金项目(31972544)。

摘　　要：为深入研究TMEM182基因在鸡体内的功能和表现,试验采用原始生殖细胞介导法,利用CRISPR/Cas9介导的HMEJ基因编辑技术,对体外培养的原始生殖细胞TMEM182基因进行敲除,以期产生TMEM182基因缺陷性腺嵌合体鸡。选择3个sgRNA靶向TMEM182基因的第二外显子,利用T7E1进行基因打靶效率检测。对体外培养的PGCs进行外源基因mCherry定点插入,引起TMEM182基因插入突变,通过流式分选获得mCherry+PGCs。将mCherry+PGCs注射到发育至2.5 d的受体鸡胚,观察外源PGCs的性腺整合情况。结果显示:TMEM182-sgRNA1的打靶效率最高;通过将sgRNA1与模板质粒共转染PGCs,得到能稳定表达红色荧光的mCherry+PGCs;孵化第6天,在受体鸡胚性腺能明显观察到发出红色荧光的PGCs,最终孵化出10只小鸡。研究表明:利用CRISPR/Cas9技术对鸡PGCs进行基因编辑,制备性腺嵌合体鸡是一种高效、稳定的制备基因编辑鸡的方法。In order to deeply investigate the function and expression of the TMEM182 gene in chickens,the experiment was conducted using a primordial germ cell-mediated assay to knockdown the TMEM182 gene in primordial germ cells cultured in vi-tro using CRISPR/Cas9-mediated HMEJ gene editing technology with the aim of generating chickens with defective gonadal chi-merism for the TMEM182 gene.Three sgRNAs were selected to target the second exon of the TMEM182 gene,and T7E1 was used to test the gene targeting efficiency.PGCs cultured in vitro were site-specific insertion with the exogenous gene mCherry,causing an insertion mutation in the TMEM182 gene,and positive cells were obtained through flow cytometry.These positive PGCs were then injected into 2.5-day-age host chicken embryos,and the integration of exogenous PGCs into the gonadal was observed.The results showed that the gene targeting efficiency of TMEM182-sgRNA1 was the highest.By co-transfecting sgRNA1 with the tem-plate plasmid into PGCs,mCherry+PGCs stably expressing red fluorescence were obtained.On the sixth day of incubation,PGCs emitting red fluorescence were clearly observed in the gonads of host chicken embryos,and eventually,ten chicks were hatched.The overall results demonstrated that CRISPR/Cas9 technology was an efficient and stable method for preparing gene-edited chickens by gene editing chicken PGCs to produce gonad chimera chickens.

关 键 词：鸡 CRISPR/Cas9 PGCS TMEM182基因 基因编辑 

分 类 号：S831.2[农业科学—畜牧学]