CRISPR/Cas9介导的Fam171a1基因敲除大鼠基因型鉴定及基因功能的初步研究

摘要

目的 通过建立Fam171a1基因敲除大鼠模型,观察基因敲除后大鼠的表型变化,探索该基因的功能.方法 采用CRISPR/Cas9打靶技术,获得Fam171a1+/-基因型杂合子.Fam171a1+/-大鼠交配获得Fam171a1-/-基因型纯合子大鼠.采用DNA Analyzer 3730XL测序技术进行基因型鉴定.对功能未知基因Fam171a1进行基本生物信息检索.检测FAM171A1蛋白在野生型大鼠(wild type,WT)和Fam171a1-/-基因型大鼠各组织的表达情况.对子代鼠20天体重、子代生育情况进行统计.采用全自动生化仪对血清生物化学指标进行检测.结果 获得了稳定遗传的Fam171a1-/-基因型大鼠.通过配笼繁殖共获得F2代125只,纯合子(Fam171a1-/-)25只、杂合子(Fam171a1+/-)71只、野生型(WT)29只,比例接近1:2:1.FAM171A1蛋白在WT大鼠肝脏等主要脏器表达,其中肺组织、脑组织和胰腺组织表达量较高,心、肝、脾和淋巴结中表达较弱,肾、胸腺和小肠组织中弱表达或不表达.同时,Fam171a1-/-基因型大鼠各组织中不表达FAM171A1蛋白.Fam171a1-/-子代大鼠出生20天体重显著低于WT大鼠(t=3.211,P=0.0017).成年Fam171a1-/-大鼠血清胆碱酯酶、总蛋白、AST和HDL水平显著高于WT(t=4.18,P<0.001;t=6.26,P<0.001;t=2.73,P=0.02;t=2.36,P=0.03).与Fam171a1+/-大鼠相比,Fam171a1-/-大鼠生育能力无显著差异(t=0.2132,P=0.8330).结论 成功获得稳定遗传的Fam171a1基因敲除大鼠,Fam171a1基因可能参与SD大鼠的生长发育、肝功能的维持及脂代谢的调控.%Objective To explore the function of Fam171a1 gene by observing the phenotype changes of the gene knockout rats. Methods Fam171a1+/- genotype rats were made by CRISPR/ Cas9 targeting technology and Fam171a1-/- genotype rat was obtained by mating two Fam171a1+/- genotype rats. Genotypes were identified by DNA Analyzer 3730XL sequencing technology. The biological function of Fam171a1 gene was explored by basic information retrieval. The expression of Fam171a1 protein in liver, brain and other major organs of WT and Fam171a1-/- rats were detected by Western blot technique. The weight of 20 days old filial rats was recorded and the serum biochemical indexes were detected by automatic biochemical analyzer.Results Stable genetic Fam171a1-/- genotype rats were obtained. Total of 125 Fam171a1+/- rats were produced,including 25 Fam171a1-/- rats, 71 Fam171a1+/- rats and 29 WT rats. FAM171A1 protein expressed in liver and other major organs especially lung, brain and pancreatic tissue, and did not express or weakly expressed in kidney, thymus and small intestine, but did not express in tissue of Fam171a1-/- rat. The average weight of Fam171a1-/- child rats was significantly lower than that of WT rats (t = 3.211, P = 0.0017), and serum CHE,TP, AST and HDL level of Fam171a1-/- adult rats were higher (t = 4.18, P < 0.001; t = 6.26, P < 0.001; t =2.73, P = 0.02; t =2.36, P = 0.03).Fertility was comparable between Fam171a1-/- and WT rats (t = 0.2132, P =0.8330). Conclusions Fam171a1 gene knockout SD rat was successfully obtained, and Fam171a1 gene may be involved in the growth and lipid metabolism of SD rats and maintaining liver function.