Abstract: Objective Constructing an adult cardiomyocyte-specific Oga knockout mouse model to study the function of endogenous OGA in the maintenance of adult cardiac homeostasis. Method Firstly, Screening the effective sgRNAs targeting the Oga coding region and and constructed them in AAV9 virus. Then AAV9 was injected into cardiomyocyte specific SpCas9 knockin mice ( α-MHC^Cas9) by intraperitoneal injection. qPCR and Western blot were performed to confirm whether Oga was successfully knockout. Finally, to study the function of Oga in cardiac homeostasis maintenance, the cardiac structure was analyzed by histology and the cardiac function was analyzed by transthoracic echocardiography. Result Two effective sgRNAs targeting Oga were identified and delivered by AAV9 to disrupt Oga gene successfully in cardiomyocytes. Consistently, O-GlcNAcylation level was significantly increased by Oga deletion. There were no significant differences in overall structure and function indicated by the histological and echocardiographic analyses. Conclusion By using the AAV9-mediated delivery of CRISPR / Cas9 system, we successfully constructed the adult cardiomyocyte- specific Oga knockout mouse model, which would be provided as an effective mouse model for studying the function and mechanism of OGA-mediated O-GlcNAcylation clearance in the maintenance of cardiac homeostasis.

Key words: CRISPR / Cas9, OGA, O-GlcNAcylation, AAV9, cardiomyocyte

摘要： 目的 利用腺相关病毒 9(AAV9)介导的 CRISPR / Cas9 系统构建成体心肌细胞特异性 Oga 基因敲除小鼠模型,来研究内源性 OGA 在成体心脏稳态维持中的功能。 方法 首先,筛选靶向 Oga 编码区的有效 sgRNA,构建包装表达有效 sgRNA 的 AAV9 病毒。 其次,建立心肌细胞特异性 SpCas9 表达小鼠 ( α-MHC^Cas9) ,并通过腹腔注射方式将 AAV9-sgOga 注射到小鼠体内。 通过 qPCR 和 Western blot 印记杂交实验检测 Oga 的表达情况,确定 Oga 敲除是否成功。 最后,通过组织学分析心脏结构,以及超声心动图分析心脏功能,来分析 Oga 对心脏稳态维持的影响。结果 筛选到 2 条可以有效靶向 Oga 编码区的 sgRNA,通过 AAV9 递送实现了 Oga 基因在心脏组织的有效敲除,且导致 O-GlcNAcylation 表达显著上升;组织学分析和超声心动图分析发现基因敲除小鼠心脏结构及功能在基础水平与对照小鼠无显著变化。 结论 利用 AAV9 介导的 CRISPR / Cas9 系统成功建立了成体心肌细胞 Oga 基因敲除的小鼠模型,为研究 OGA 介导的 O-GlcNAcylation 清除在心脏稳态维持中的功能和机制提供了有效的小鼠模型。

关键词: CRISPR / Cas9, OGA, O-GlcNAcylation, AAV9, 心肌细胞