利用shRNA慢病毒高效敲降雄性小鼠睾丸靶向基因
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南京医科大学生殖医学国家重点实验室

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国家自然科学基金项目(面上项目,重点项目,重大项目)


Application of shRNA-mediated lentivirus for knockdown of endogenous genes in mouse testis
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State Key Laboratory of Reproductive Medicine,Nanjing Medical University,Nanjing,Jiangsu

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    摘要:

    目的:建立快速、高效的制备小鼠睾丸基因敲降的平台。方法:设计和构建靶向睾丸特异性表达基因Sox30转录本的shRNA表达载体(pSliencer:GFP:shSox30)及其对照质粒(pSliencer:GFP:shScramble),与慢病毒颗粒包装后通过网微注射转导至24天小鼠睾丸生精小管管腔,利用免疫荧光等技术方法检测慢病毒敲降鼠体内靶向基因的效率及shRNA敲降Sox30后对生殖细胞发育的影响。结果:慢病毒处理敲降后,相比于对照组小鼠,shSox30病毒敲降组小鼠睾丸组织中SOX30蛋白表达水平显著下调,生精管腔中圆形精子发育阻滞在2~3步,VII-VIII期生精管腔中无长型精子。结论:shRNA慢病毒有效降低小鼠睾丸靶向基因的表达水平,在制备睾丸特定基因敲降动物模型中具有巨大优势。

    Abstract:

    Objective: This study aims to establish an efficient and fast method for in vivo knockdown of endogenous genes in mouse testis. Methods: We designed and constructed the lentiviral vector (pSliencer:GFP:shSox30) along with the control vector (pSliencer:GFP:shScramble), and prepared high-titer lentiviral particles. These lentiviral particles were microinjected into the mouse testis at postnatal day 24. Immunofluorescence and histological analysis was performed to evaluate the functional consequences in mouse testis upon Sox30 knock down. Results: After the shSox30 lentiviral particles treatment, SOX30 protein was substantially decreased in mouse testis. Immunofluorescence of testis sections revealed that many spermatids were arrested at step 2-3. Histological examination of testis sections revealed that elongated spermatids were absent in seminiferous tubules at stage VII-VIII. Conclusion: shRNA-mediated lentivirus effectively decreases the expression level of target genes in mouse testis, providing a useful platform to knockdown endogenous genes in vivo.

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  • 收稿日期:2022-11-29
  • 最后修改日期:2023-03-31
  • 录用日期:2023-05-23
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