利用CRISPR/Cas9系统敲除小鼠Mlk3基因探讨其对血压的影响
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国家自然科学基金(91939106,81770470);北京市自然科学基金(7192032)


Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure
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    摘要:

    为探讨混合谱系激酶3 (mixed lineage kinase 3, Mlk3)基因敲除(knockout, KO)对小鼠血压的影响,采用CRISPR/Cas9系统构建Mlk3基因敲除(Mlk3 knockout,Mlk3KO)小鼠模型。T7核酸内切酶I (T7 endonuclease I, T7E1)酶切法验证向导RNA (small guide RNA, sgRNA)的活性。体外转录CRISPR/Cas9 mRNA及sgRNA,显微注射至受精卵并移植入假孕母鼠。聚合酶链式反应(polymerase chain reaction, PCR) 及DNA测序检测基因型。实时荧光定量PCR (Real-time PCR, RT-PCR)检测Mlk3 mRNA表达,Western blotting及免疫荧光检测Mlk3蛋白表达。尾套法监测小鼠血压。免疫组化及Western blotting检测小鼠主动脉肌球蛋白轻链(myosin light chain, MLC)磷酸化水平。PCR基因型鉴定及DNA测序显示Mlk3敲除成功,且Mlk3敲除小鼠未检测到Mlk3蛋白表达,证实CRISPR/Cas9系统成功构建了Mlk3敲除小鼠。Mlk3敲除小鼠在基础状态下血压显著高于同笼对照,且Mlk3敲除小鼠主动脉平滑肌层MLC的磷酸化高于对照组,说明Mlk3具有抗高血压的内源性保护作用。本研究为探讨蛋白激酶Mlk3抗高血压及抗高血压诱导心血管不良重塑的作用机制提供了理想的动物模型。

    Abstract:

    To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

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高诗娟,方光明,张燕红,杜杰. 利用CRISPR/Cas9系统敲除小鼠Mlk3基因探讨其对血压的影响[J]. 生物工程学报, 2023, 39(4): 1644-1654

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  • 收稿日期:2022-12-22
  • 最后修改日期:
  • 录用日期:2023-02-16
  • 在线发布日期: 2023-04-14
  • 出版日期: 2023-04-25
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