斑马鱼gpr112a基因敲除品系的构建  

Construction of Zebrafish gpr112a Gene Knockout Line

作　　者：孙鲁宁 杨博宇 刘玲[1,2] 朱俊伟 杨添乐 彭政 郑澜[3] 谢华平[1,2,4] SUN Luning;YANG Boyu;LIU Ling;ZHU Junwei;YANG Tianle;PENG Zheng;ZHENG Lan;XIE Huaping(Hunan Normal University Hunan Key Laboratory of Animal Intestinal Function Regulation,Changsha 410081;Hunan Normal University Laboratory of Animal Nutrition and Human Health,Changsha 410081;Hunan Normal University Hunan Key Laboratory of Physical Fitness and Sports Rehabilitation,Changsha 410081;Hunan Normal University State Key Laboratory of Freshwater Fish Developmental Biology,Changsha 410081)

机构地区：[1]湖南师范大学动物肠道功能调控湖南省重点实验室,长沙410081 [2]湖南师范大学动物营养与人体健康实验室,长沙410081 [3]湖南师范大学体适能与运动康复湖南省重点实验室,长沙410081 [4]湖南师范大学淡水鱼类发育生物学国家重点实验室,长沙410081

出　　处：《激光生物学报》2022年第6期526-532,共7页Acta Laser Biology Sinica

基　　金：国家自然科学基金项目(31970504);湖南省科技领军人才项目(2019RS3022)。

摘　　要：GPR112蛋白作为黏附类G蛋白偶联受体(aGPCRs)家族的一员,由GPR112基因编码。它含有7个跨膜结构域,与G蛋白相互作用行使其功能。斑马鱼作为研究脊椎动物发育的重要模式生物,其基因组中含有GPR112的同源基因gpr112a,且在幼鱼和成鱼中均有表达。为了研究GPR112在脊椎动物生存和发育中的作用,利用CRISPR-Cas9技术构建了gpr112a基因敲除斑马鱼品系。首先,合成用于靶向gpr112a基因第二外显子的一对sgRNAs,经显微注射得到F0代嵌合体斑马鱼。然后,对嵌合体与野生型斑马鱼杂交所得的F1代斑马鱼进行基因型鉴定,筛选gpr112a突变杂合子,并对其gpr112a突变等位基因进行Sanger测序,以确定gpr112a基因敲除品系的建立。之后,gpr112a突变杂合子斑马鱼自交,获得gpr112a纯合子突变斑马鱼。经显微成像并观察发现,受精后7 dpf的gpr112a突变纯合子斑马鱼并没有出现与野生型差异明显的表型,这可能是斑马鱼机体的代偿机制所致。此研究为探究GPR112基因在脊椎动物生存和发育中的作用开辟了道路。GPR112 protein, as a member of adhesion class G protein-coupled receptors family(aGPCRs), is encoded by GPR112 gene. It contains seven transmembrane domains and interacts with G protein to perform its functions. Zebrafish is an important model organism for studying the development of vertebrates. Its genome contains the homologous gene GPR1112a of GPR112, which is expressed in both juvenile and adult fish. To study the role of GPR112 in vertebrate survival and development, CRISPR-Cas9 technology was utilized to construct a gpr112a knockout zebrafish line. First, a pair of sgRNAs targeting the second exon of the gpr112a gene were synthesized and microinjected to obtain F0 generation chimeric zebrafish. Next, the F1 generation zebrafish obtained by mating the chimeras with wild-type zebrafish were genotyped. The gpr112a mutant heterozygotes were screened and the gpr112a mutant alleles were subjected to Sanger sequencing to determine the establishment of gpr112a knockout lines. The gpr112a mutant heterozygous zebrafish were then selfed to obtain gpr112a mutant homozygous zebrafish. After microscopic imaging and observation, the zebrafish homozygous for the gpr112a mutation at 7 days post fertilization did not show a phenotype that was significantly different from that of the wild type, which may be caused by the compensatory mechanism of the zebrafish body. This study laid a foundation for exploring the role of GPR112 gene in vertebrate survival and development.

关 键 词：斑马鱼 gpr112a CRISPR-Cas9 基因敲除 代偿机制 

分 类 号：Q341[生物学—遗传学] Q812