CAJ | 학술논문

目的：为研究胰岛素受体底物1（Irs1）基因与代谢病之间的关系，我们利用CRISPR/Cas9系统敲除大鼠Irs1基因，为研究代谢病提供基因敲除大鼠。方法针对Irs1第一外显子，设计CRISPR/Cas9作用靶点，构建sgRNA表达质粒。利用T7 RNA聚合酶体外转录sgRNA和Cas9。将Cas9 mRNA和sgRNA混合物注射入SD大鼠的受精卵中，实现靶基因敲除。用T7 EN1实验初步检测靶基因的修饰情况，再经过测序分析确定突变。结果获得了5个在Irs1基因突变的首建鼠，突变效率为83％。结论得到了稳定遗传的Irs1基因敲除大鼠。
목적：위연구이도소수체저물1（Irs1）기인여대사병지간적관계，아문이용CRISPR/Cas9계통고제대서Irs1기인，위연구대사병제공기인고제대서。방법침대Irs1제일외현자，설계CRISPR/Cas9작용파점，구건sgRNA표체질립。이용T7 RNA취합매체외전록sgRNA화Cas9。장Cas9 mRNA화sgRNA혼합물주사입SD대서적수정란중，실현파기인고제。용T7 EN1실험초보검측파기인적수식정황，재경과측서분석학정돌변。결과획득료5개재Irs1기인돌변적수건서，돌변효솔위83％。결론득도료은정유전적Irs1기인고제대서。
Objective To study the relationship of insulin receptor substrate-1 (Irs1) and metabolic disease, we generated Irs1 gene knockout rat by CRISPR/Cas9 system.Methods Two sgRNA targeting sites were designed for Irs1 targeting.The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro.Cas9 mRNA and sgRNA mixtures were pooled and microinjected into one-cell fertilized eggs of SD rats to generate rats with targeted mutation .Results Five rats with the mutations were detected with the efficiency of 83%.Conclusion The Irs1 gene knockout rats generated in this study can be transmitted by germline .