利用shRNA慢病毒高效敲降雄性小鼠睾丸基因  

Application of shRNA⁃mediated lentivirus for knockdown of endogenous genes in mouse testis

作　　者：龚洁[1] 卜治文 徐晨 叶岚 谢婕 GONG Jie;BU Zhiwen;XU Chen;YE Lan;XIE Jie(State Key Laboratory of Reproductive Medicine,Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京医科大学生殖医学国家重点实验室,江苏南京211166

出　　处：《南京医科大学学报（自然科学版）》2023年第6期749-755,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金项目(31871503,32070843,2022YFC2703501)。

摘　　要：目的:建立快速、高效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出生24 d小鼠睾丸生精小管管腔,利用免疫荧光等检测慢病毒敲降鼠体内靶向基因的效率及敲降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。Objective:The current study aims to establish an efficient and fast method for in vivo knockdown of endogenous genes in mouse testis.Methods:Lentiviral vector(pSliencer⁃GFP⁃shSox30)along with the control vector(pSliencer⁃GFP⁃shScramble)were designed and constructed.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 knockdown.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 stageⅦ⁃Ⅷ.Conclusion:The shRNA⁃mediated lentivirus effectively decreases the expression level of target genes in mouse testis,providing a useful platform to knockdown endogenous genes in vivo.

关 键 词：RNA干扰 动物模型 Sox30 小鼠 睾丸 

分 类 号：R394.33[医药卫生—医学遗传学]