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CRISPR/Cas9技术在构建荧光标记柔嫩艾美耳球虫β-微管蛋白敲入虫株上的应用

文献类型: 中文期刊

作者: 肖琴 1 ; 曲自刚 1 ; 李文卉 1 ; 张念章 1 ; 孙晓林 1 ; 付宝权 1 ;

作者机构: 1.甘肃农业大学动物医学院;中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心

关键词: 柔嫩艾美耳球虫;CRISPR/Cas9;基因敲入;β-tubulin

期刊名称: 中国兽医科学

ISSN: 1673-4696

年卷期: 2024 年 54 卷 06 期

页码: 742-748

收录情况: 北大核心 ; CSCD

摘要: 为评价弓形虫CRISPR/Cas9质粒在柔嫩艾美耳球虫基因敲入中的应用,以柔嫩艾美耳球虫Etβ-tubulin为目标基因,根据Etβ-tubulin基因序列设计向导RNA (g RNA),构建含g RNA的基因编辑质粒及含有目标基因和m Cherry基因的同源重组质粒;将含g RNA的基因编辑质粒和含有目标基因、DHFR基因和m Cherry基因的同源重组质粒共同转染柔嫩艾美耳球虫子孢子,将转染后的子孢子接种雏鸡泄殖腔,并在饲料中添加乙胺嘧啶进行筛选,得到表达外源荧光蛋白的标记球虫虫株。挑取单卵囊进行扩繁,并通过PCR和荧光检测方法分别从基因水平和蛋白水平上对标记虫株进行了鉴定。结果显示通过单卵囊筛选和鉴定,成功获得了1株荧光标记β-微管蛋白的柔嫩艾美耳球虫虫株。结果表明,CRISPR/Cas9技术可应用到柔嫩艾美耳球虫的基因敲入研究,同时也为进一步研究Etβ-tubulin在虫体中的功能奠定了基础。

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