摘要
目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6J小鼠,针对miR-551b外显子1区域,设计导向RNA(guide RNA,gRNA),构建Cas9载体质粒,将体外转录的Cas9 RNA及gRNA显微注射入小鼠的受精卵并体外培养。将培养合格的胚胎移植到代孕小鼠的输卵管中,待小鼠生育后得到F0代小鼠,使用基因测序确定基因敲除情况,与野生型小鼠繁育后,得到F1代杂合小鼠,F1代小鼠经自交繁育获得F2代小鼠,F3代小鼠由F2代纯合小鼠自交获得,采用电泳鉴定小鼠基因型,RT-PCR检测F3代小鼠组织miR-551b的表达。结果:利用CRISPR/Cas9技术构建模型小鼠得到F0代小鼠,通过测序筛选出缺失目标序列的F0代杂合子小鼠。与WT小鼠繁育后,琼脂糖凝胶电泳及测序筛选出F1代杂合小鼠,同样的方法鉴定并获得F2、F3代基因敲除小鼠,获取F3代纯合小鼠的心脏及下腔静脉样本,RT-PCR结果证实F3代纯合小鼠miR551b表达明显低于WT小鼠(P<0.05),成功敲除miR-551b基因。结论:通过CRISPR-Cas9技术成功构建miR⁃155基因敲除小鼠模型并稳定遗传,为进一步研究提供了有利条件。
Objective:To construct a mouse model of miR-551b gene knockout using clustered regularly interspaced short palindromic repeat associated protein 9(CRISPR-Cas9)technique.Methods:C57BL/6J mice were selected to construct the model.According miR-551b exon1 sequence,we designed guide RNAs.The Cas9 RNA and gRNA transcribed in vitro were microinjected into the fertilized eggs of mice and cultured.The qualified embryos were transferred to surrogate mice,F0 generation mice were obtained,and gene sequencing was used to detect the gene knockout situation.After breeding with WT mice,F1 generation heterozygous mice were obtained,and F2 generation mice were obtained by self-cross breeding of F1 generation mice.The F3 mice were obtained by self-crossed from the F2 homozygous mice.F2,F3 homozygous mice were selected according to electrophoresis results,and the expression of miR551b in heart and inferior vena cava of F3 homozygous mice was detected by RT-PCR.Results:Model mice were constructed using CRISPR-Cas9 technology to obtain F0 generation mice,and F0 mice missing target sequence were detected by sequencing.After breeding with WT mice,F1 heterozygous mice were screened according to PCR electrophoresis results,F2,F3 homozygous gene knockout mice were screened by the same method,and the heart and inferior vena cava samples of F3 homozygous mice were obtained.RT-PCR results confirmed that the expression of miR-551b in F3 homozygous mice was significantly lower than WT mice(P<0.05).Conclusions:The mouse model of miR-155 gene knockout was successfully constructed by CRISPR-Cas9 technique and its inheritance was stable,providing favorable conditions for further research.
作者
张魁
黄柱辉
周宁
曹剑
付威
郑居兵
董然
ZHANG Kui;HUANG Zhuhui;ZHOU Ning;CAO Jian;FU Wei;ZHENG Jubing;DONG Ran(Department of Cardiac Surgery,Beijing Anzhen Hospital,Capital Medical University,Beijing Institute of Heart,Lung and Blood Vessel Diseases,Beijing 100029,China)
出处
《心肺血管病杂志》
CAS
2023年第7期734-738,共5页
Journal of Cardiovascular and Pulmonary Diseases
基金
国家自然科学基金(81570373,81770412)。
