基于CRISPR/Cas9构建血管平滑肌细胞特异性Mrjp 1基因敲入小鼠  

Generation of Mrjp 1 Gene Knock-in Mice with Specific Expression in Vascular Smooth Muscle Cells via CRISPR/Cas9 Strategy

作　　者：沙芳芳 范沛[1,2] 杨培长 张璐 李建科[2] SHA Fangfang;FAN Pei;YANG Peichang;ZHANG Lu;LI Jianke(School of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China;Institute of Apicultural Research,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]河南工业大学生物工程学院,郑州450001 [2]中国农业科学院蜜蜂研究所,北京100193

出　　处：《中国畜牧兽医》2023年第11期4392-4402,共11页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金资助项目(32070742);农业农村部授粉昆虫生物学重点实验室开放基金资助项目(2018MFNZS02)。

摘　　要：【目的】探究蜂王浆中含量最高的活性分子——蜂王浆主蛋白1(MRJP1)基因敲入血管平滑肌细胞后,在体内水平上是否具有调节小鼠血压的功能。【方法】利用CRISPR/Cas9技术,将可识别小鼠Hipp11位点的gRNA、Cas9和包含Sm22α启动子+Mrjp 1 cDNA的载体共显微注射小鼠受精卵,制备在血管平滑肌细胞中特异性表达的Mrjp 1基因定点敲入小鼠,并验证敲入位点的正确性。小鼠背部皮下包埋微渗透压缓释泵,持续将血管紧张素Ⅱ(AngⅡ)诱导野生型(WT)和纯合子(Mrjp 1^(+/-))小鼠,比较不同时间收缩压和舒张压,以及胸主动脉中膜面积/管腔面积(Media/Lumen area)比值的差异。【结果】F0代嵌合体小鼠与野生型交配,产生杂合子(Mrjp 1^(+/-))小鼠,对Mrjp 1^(+/-)小鼠敲入位点同源重组片段的PCR扩增、测序和Southern blotting检测结果显示,Mrjp 1基因成功整合到小鼠染色体Hipp11位点。Mrjp 1^(+/-)小鼠之间交配产生WT、Mrjp 1^(+/-)和Mrjp 1^(+/-)小鼠,Mrjp 1基因在胸主动脉中能够顺利转录和翻译。持续给予AngⅡ,Mrjp 1^(+/-)小鼠收缩压和舒张压升高趋势整体低于WT小鼠,收缩压在第3、6、9、12天差异显著(P<0.05),舒张压在第3(P<0.05)、6(P<0.01)天差异显著;经AngⅡ刺激后,M rjp 1^(+/-)小鼠胸主动脉Media/Lumen area比值亦显著低于WT小鼠(P<0.05)。【结论】Mrjp 1基因特异性敲入小鼠血管平滑肌细胞,可在体内水平上抑制AngⅡ诱导的高血压,为深入了解MRJP1蛋白功能及精准开发蜂王浆提供了重要的理论依据。【Objective】The aim of this study was to disclose whether the introduction of major royal jelly protein 1(MRJP1)to vascular smooth muscle cells(VSMCs)could regulate mice blood pressure in vivo.【Method】Based on the CRISPR/Cas9 strategy,the gRNA to Hipp11 site,Cas9,and donor vector containing honeybee Mrjp1 cDNA with the upstream Sm22αpromoter were co-injected to embryos to produce the Mrjp 1 gene knock-in mice that Mrjp 1 gene specifically expressed in VSMCs,followed by the verification of knock-in correctness.The systolic and diastolic blood pressures of different days and Media/Lumen area ratios of thoracic aorta for wild type(WT)and homozygous(Mrjp 1^(+/-))mice with continuous angiotensinⅡ(AngⅡ)infusion by mini-osmotic pumps implanted subcutaneously on the back were compared.【Result】The heterozygous(Mrjp 1^(+/-))mice were generated by crossing F0 chimeric with WT mice.PCR amplification and sequencing,as well as Southern blotting,for homology fragments showed that Mrjp 1 gene was successfully integrated into the Hipp11 site of mouse chromosome.The WT,Mrjp 1^(+/-)and Mrjp 1^(+/-)mice were generated through Mrjp 1^(+/-)mating.Mrjp 1 gene was able to transcript and translate in thoracic aorta.Continuously induced by AngⅡ,the blood pressure,including systolic and diastolic,of the Mrjp 1^(+/-)mice showed the lower trend than that of the WT.To be exact,the systolic blood pressure at the days of 3,6,9 and 12 were significantly different(P<0.05),and the diastolic blood pressure at the days of 3(P<0.05)and 6(P<0.01)were significantly different.After the treatment of AngⅡ,the Media/Lumen area ratio of thoracic aorta of Mrjp 1^(+/-)mice was also significantly lower than that of the WT(P<0.05).【Conclusion】The specific knock-in of Mrjp 1 gene to VSMCs inhibited the mice blood pressure stimulated by AngⅡin vivo.The current work would cast light on the in-depth elucidation of MRJP1 function and the precise utilization of royal jelly.

关 键 词：CRISPR/Cas9 小鼠 基因敲入 蜂王浆主蛋白1 血压调节 

分 类 号：S896.3[农业科学—特种经济动物饲养]