利用CRISPR/Cas9技术构建RAG1基因敲除免疫缺陷猪模型的研究  

Construction of immunodeficiency pig model with RAG1 gene knockout using CRISPR/Cas9 technology

作　　者：王清未 李振阳 张效生[2,3,4] 张林林 WANG Qingwei;LI Zhenyang;ZHANG Xiaosheng;ZHANG Linlin(College of Animal Science and Veterinary Medicine,Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,Tianjin Agricultural University,Tianjin 300384,China;Institute of Animal Science and Veterinary,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China;Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology,Tianjin 300381,China;Tianjin Engineering Research Center of Animal Healthy Farming,Tianjin 300381,China)

机构地区：[1]天津农学院动物科学与动物医学学院天津市农业动物繁育与健康养殖重点实验室,天津300384 [2]天津市农业科学院畜牧兽医研究所,天津300381 [3]天津市畜禽分子育种与生物技术重点实验室,天津300384 [4]天津市畜禽健康养殖工程技术中心,天津300384

出　　处：《黑龙江畜牧兽医》2024年第9期1-7,114,115,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(32071456);天津农学院研究生科研创新项目(2021XY039)。

摘　　要：为了利用显微注射一步法构建RAG1基因敲除的免疫缺陷猪模型,试验利用CRISPR/Cas9技术针对猪的RAG1基因设计并筛选出高效的sgRNA,与体外转录的Cas9 mRNA按照4∶6比例混合后显微注射到猪体外受精的受精卵中,随后将基因编辑的受精卵移植到受体母猪输卵管内;仔猪出生后采集耳缘皮肤组织提取基因组进行PCR扩增、T7核酸内切酶Ⅰ酶切鉴定,以及利用Sanger法测序鉴定编辑后基因型;监测基因敲除的免疫缺陷仔猪在普通环境的生存周期;对死亡的仔猪进行剖检,采集胸腺和脾脏等免疫器官进行组织学分析。结果表明:通过在猪成纤维细胞系水平上进行RAG1基因敲除效率的筛选,筛选出针对猪RAG1基因第2外显子高效敲除的sgRNA序列,通过对猪受精卵胞质显微注射sgRNA和Cas9 mRNA混合物,获得2头RAG1基因敲除免疫缺陷仔猪,经鉴定发现RAG1基因的第2外显子碱基序列中有1 bp插入,造成移码突变,破坏了RAG1基因功能,致使仔猪免疫系统缺陷。出生的2头免疫缺陷仔猪在普通饲养环境的存活时间分别为19 d和33 d。RAG1基因敲除的免疫缺陷仔猪胸腺缺失,脾脏发育不良,无法在正常饲养环境中长时间存活。脾脏结构紊乱,出现炎症细胞浸润等免疫系统异常。说明通过CRISPR/Cas9技术进行显微注射可获得RAG1基因敲除的免疫缺陷猪模型。In order to construct an immunodeficient pig model with RAG1 gene knockout by microinjection in one step,CRISPR/Cas9technology was used to design and screen out efficient sgRNA for the RAG1 gene of pigs.It was mixed with Cas9 mRNA transcribed in vitro in a4∶6 ratio and microinjected into the fertilized egg of pig in vitro fertilization,and then the gene-edited fertilized egg was transplanted into the uterus of the recipient sow.After the piglets were born,the skin tissue of the ear margin was collected and the genome was extracted for PCR amplification,T7 endonuclease I enzymatic identification,and identification of the edited genotype by Sanger sequencing.The survival cycle of immunodeficient piglets with gene knockout was monitored in normal environment.Immune organs such as thymus and spleen of dead gene-edited piglets were collected for histological analysis.The results showed that the knockout efficiency of RAG1 gene was screened at the level of porcine fibroblast cell line.The sgRNA sequences for the high efficiency knockout of exon 2 of porcine RAG1 gene were screened.Two RAG1knockout immunodeficient piglets were obtained by microinjection of sgRNA and Cas9 mRNA mixture into the recipient oocyte.The insertion of1 bp into the second exon base sequence of RAG1 gene resulted in frameshift mutation,which destroyed the function of RAG1 gene and resulted in immune system defect of piglets.The survival time of the two immunodeficient piglets born in the ordinary breeding environment was 19 d and33 d,respectively.Pathological sections and tissue morphology tests were performed on the dead piglets.The phenotypes of the immunodeficient pigs with RAG1 gene knock-out showed abnormalities such as athymism and dysplasia of the spleen,and they could not survive in the normal breeding environment.Spleen structure disorder,inflammatory cell infiltration and other immune system abnormalities.These results indicated that RAG1 knockout immunodeficient pig models could be obtained by microinjection with CRISPR/Cas9 technolog

关 键 词：免疫缺陷动物 CRISPR/Cas9 猪 RAG1 sgRNA 

分 类 号：S828[农业科学—畜牧学]