基于CRISPR/Cas9构建血管平滑肌细胞特异性Mrjp1基因敲入小鼠

doi:10.16431/j.cnki.1671-7236.2023.11.008

摘要/Abstract

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+Mrjp1 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Mrjp1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（Mrjp1^+/+）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（Mrjp1^+/-）小鼠，对Mrjp1^+/-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，Mrjp1基因成功整合到小鼠染色体Hipp11位点。Mrjp1^+/-小鼠之间交配产生WT、Mrjp1^+/-和Mrjp1^+/+小鼠，Mrjp1基因在胸主动脉中能够顺利转录和翻译。持续给予AngⅡ，Mrjp1^+/+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，Mrjp1^+/+小鼠胸主动脉Media/Lumen area比值亦显著低于WT小鼠（P<0.05）。【结论】Mrjp1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。

关键词: CRISPR/Cas9; 小鼠; 基因敲入; 蜂王浆主蛋白1; 血压调节

Abstract: 【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 Sm22α promoter were co-injected to embryos to produce the Mrjp1 gene knock-in mice that Mrjp1 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 (Mrjp1^+/+) mice with continuous angiotensinⅡ(AngⅡ) infusion by mini-osmotic pumps implanted subcutaneously on the back were compared.【Result】 The heterozygous (Mrjp1^+/-) mice were generated by crossing F0 chimeric with WT mice.PCR amplification and sequencing, as well as Southern blotting, for homology fragments showed that Mrjp1 gene was successfully integrated into the Hipp11 site of mouse chromosome.The WT, Mrjp1^+/- and Mrjp1^+/+mice were generated through Mrjp1^+/-mating.Mrjp1 gene was able to transcript and translate in thoracic aorta.Continuously induced by AngⅡ, the blood pressure, including systolic and diastolic, of the Mrjp1^+/+ 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 Mrjp1^+/+ mice was also significantly lower than that of the WT (P<0.05). 【Conclusion】 The specific knock-in of Mrjp1 gene to VSMCs inhibited the mice blood pressure stimulated by AngⅡ in vivo.The current work would cast light on the in-depth elucidation of MRJP1 function and the precise utilization of royal jelly.

Key words: CRISPR/Cas9; mice; gene knock-in; major royal jelly protein 1; blood pressure regulation

中图分类号:

S896.3

沙芳芳, 范沛, 杨培长, 张璐, 李建科. 基于CRISPR/Cas9构建血管平滑肌细胞特异性Mrjp1基因敲入小鼠[J]. 中国畜牧兽医, 2023, 50(11): 4392-4402.

SHA Fangfang, FAN Pei, YANG Peichang, ZHANG Lu, LI Jianke. Generation of Mrjp1 Gene Knock-in Mice with Specific Expression in Vascular Smooth Muscle Cells via CRISPR/Cas9 Strategy[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4392-4402.

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