Method for establishing CYP2C11 gene knockout rat model

一种建立cyp2c11基因敲除大鼠模型的方法

Abstract

The invention relates to a method for establishing a CYP2C11 gene knockout rat model, and belongs to the technical field of transgenosis. The method comprises the steps of firstly determining a target point of a to-be-knocked gene and designing and synthesizing a primer sequence of the gene; inserting the primer sequence into a Cas9-gRNA-Bsal carrier subjected to enzyme digestion and performing amplification to obtain a target point sgRNA with a T7 promoter sequence; after in vitro transcription and purification, performing activity detection; micro-injecting active sgRNA and Cas9 RNA into a rat single-cell embryo to obtain a Founder rat; and after screening out a CYP2C11 gene knockout rat heterozygote, performing mating to obtain a homozygote individual, namely, a CYP2C11 gene knockout rat. The invention provides a brand-new preparation method replacing a mouse model with a P450 gene knockout rat model, so that a gene knockout technology is really fused in non-clinical safety evaluation of drugs and early toxicity of candidate drugs can be discovered.
本发明涉及一种建立CYP2C11基因敲除大鼠模型的方法,属于转基因技术领域;本发明首先确定待敲除基因的靶位点并设计合成其引物序列;插入经酶切好的Cas9-gRNA-Bsal载体中,扩增得到带有T7启动子序列的靶位点sgRNA;体外转录并纯化后检测其活性;将有活性的sgRNA和Cas9?RNA显微注射入大鼠单细胞胚,获得Founder大鼠;筛选出CYP2C11基因敲除大鼠杂合子后交配得到纯合子个体,即CYP2C11基因敲除大鼠;本发明提供全新的用P450基因敲除大鼠模型代替小鼠模型的制备方法,使基因敲除技术真正融入药物的非临床安全评价,有利于候选药物的早期毒性的发现。

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