利用CRISPR/CAS9技术构建山羊乳腺上皮细胞TPH1基因敲除细胞系  

Construction of TPH1 Knockout Goat(Capra hircus) Mammary Epithelial Cell Lines by CRISPR/CAS9 System

作　　者：张志飞 田慧彬 骆琛 柏亦陈 罗军[1] 郑惠玲[1] ZHANG Zhi-Fei;TIAN Hui-Bin;LUO Chen;BAI Yi-Chen;LUO Jun;ZHENG Hui-Ling(College of Animal Science and Technology,Northwest A＆F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,杨陵712100

出　　处：《农业生物技术学报》2018年第8期1431-1439,共9页Journal of Agricultural Biotechnology

基　　金：国家自然基金面上项目(No.31572368)

摘　　要：五羟色胺(5-hydroxytryphtamine,5-HT)是机体重要的单胺类神经递质,广泛分布于动物体各组织器官并参与机体多种生理功能。色氨酸羟化酶(tryptophan hydroxylase,TPH)1是5-HT合成的限速酶,在5-HT调控机体代谢过程中起关键作用。本研究旨在利用CRISPR/CAS9技术建立稳定敲除TPH1基因的山羊(Capra hircus)乳腺上皮细胞系,为研究山羊乳腺中5-HT调控山羊乳腺上皮细胞(goat mammary epithelial cells,GMEC)钙离子代谢的分子机理提供实验材料。首先根据Gen Bank中山羊TPH1基因序列(Gen Bank No.:102184739),设计靶向TPH1基因第一外显子的单导链RNA(single guide RNA,sgRNA),用pX458和pX459分别构建重组真核表达载体pX458-sgRNA和pX459-sgRNA,将重组质粒转染山羊乳腺上皮细胞,使用嘌呤霉素(1μg/mL)进行阳性细胞筛选,挑取单克隆细胞进行培养,用Western blot、T7E1酶切、基因组DNA测序等方法鉴定基因敲除效果。结果显示:通过CRISPR/CAS9技术成功在TPH1基因第一外显子打靶产生10 bp碱基缺失。本研究成功构建并筛选获得了TPH1基因稳定敲除的单克隆山羊乳腺上皮细胞系,为5-HT调控乳腺生理的功能研究提供了重要材料。5-hydroxytryptamine （5-HT） is essential to stimulate skeletal calcium reabsorption for milk synthesis by the mammary gland. Tryptophan hydroxylase （TPH1） is the rate-limiting enzyme in peripheral 5-hydroxytryphtamine （5-HT） synthesis and thus plays an essential role in 5-HT metabolism. Objective of this study is to generate stable TPH1 gene knockout goat （Capra hircus） mammary epithelial cell line by CRISPR/Cas9 system, which could be an important material for exploring functions of 5-HT and calcium circulatory metabolism in goat mammary glands. Firstly, single guide RNA （sgRNA） sequences were designed based on the sequence of TPH1 （GenBank No.： 102184739） and were inserted into pX458 and pX459 vectors, respectively. Then goat mammary epithelial cells （GMECs） were transfected with pCas-guide vectors and puromycin （1 μg/mL） was used for screening positive cells. Finally, Western blot and DNA sequencing were used for identifying if the TPH1 gene of the GMECs had been knocked out. In this study, 3 pairs of pCAS-sgRNA vectors targeting TPH1 gene exon 1 of dairy goat were designed and constructed by using CRISPR/CAS9 technique. The transfection efficiency reached 20%; Single cell line px459-sg2-SC5 was screened by puromycin and the editing efficiency was 32%; DNA sequencing result showed that there were 10 bp base deletion at sg2 targeting site. Protein expression of TPH1 was blocked. Taken together, goat mammary epithelial cell line with stable knockout of TPH1 gene was successfully obtained, which would provide important materials for the study of the regulation of mammary gland physiology by 5-HT.

关 键 词：CRISPR/CAS9 色氨酸羟化酶(TPH) 基因敲除 山羊乳腺上皮细胞(GMEC) 

分 类 号：S827[农业科学—畜牧学]