目的探讨含SUN(Sad,UNC-84)结构域的人睾丸蛋白SPAG4L(sperm-associated antigen 4 like)与具有KASH(Klarsicht,ANC-1 and Syne homology)结构域的核膜血影重复蛋白3(nuclear envelop spectrin repeat proteins 3,Nesprin-3)之间的相互...目的探讨含SUN(Sad,UNC-84)结构域的人睾丸蛋白SPAG4L(sperm-associated antigen 4 like)与具有KASH(Klarsicht,ANC-1 and Syne homology)结构域的核膜血影重复蛋白3(nuclear envelop spectrin repeat proteins 3,Nesprin-3)之间的相互作用。方法用生物信息学方法对SPAG4L蛋白进行分析,通过体外转染实验,观察SPAG4L的亚细胞定位;并用免疫荧光技术、免疫共沉淀和双分子荧光互补实验检测SPAG4L是否与KASH结构域蛋白Nesprin-3存在相互作用。结果生物信息学分析结果表明,SPAG4L蛋白具有跨膜结构;亚细胞定位结果发现,SPAG4L蛋白定位于核膜和胞浆;免疫荧光、免疫共沉淀和双分子荧光互补实验结果表明,SPAG4L蛋白与Nesprin-3蛋白质相互作用,形成LINC(linkers of the nucleoskeleton to the cytoskeleton)复合物。结论 SPAG4L与Nesprin-3能够相互作用,形成LINC复合物,对了解SPAG4L蛋白在精子发生中的作用具有重要的意义。展开更多
The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in liv...The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels.In this study,we combined the CRISPR-RfxCas13d system with spermlike stem cell-mediated semi-cloning techniques,which enabled the suppressed expression of different RNA species.This approach was employed to interfere with the expression of three types of RNA molecules:Sfmbt2 mRNA,Fendrr lncRNA,and circMan1a2(2,3,4,5,6).The results confirmed the critical roles of these RNAs in embryonic development,as their loss led to observable phenotypes,including embryonic lethality,delayed embryonic development,and embryo resorption.In summary,our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.展开更多
在基因编辑领域,以CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)为代表的技术的广泛应用,使得科学家能快速、高效、精确地对靶基因序列进行遗传改造。但是,由于包括人类在内...在基因编辑领域,以CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)为代表的技术的广泛应用,使得科学家能快速、高效、精确地对靶基因序列进行遗传改造。但是,由于包括人类在内的真核细胞二倍性的特点,要获得纯合编辑的细胞系和模式动物仍需大量的时间和人力成本。类精子干细胞(DKO-AG-haESCs)系统的建立及其与CRISPR-Cas9技术的结合则可以快速地在基因、染色体等水平上进行任意遗传操作并转化为动物个体,实现疾病模拟。本文将对类精子干细胞介导的遗传改造进行总结和讨论。展开更多
文摘目的探讨含SUN(Sad,UNC-84)结构域的人睾丸蛋白SPAG4L(sperm-associated antigen 4 like)与具有KASH(Klarsicht,ANC-1 and Syne homology)结构域的核膜血影重复蛋白3(nuclear envelop spectrin repeat proteins 3,Nesprin-3)之间的相互作用。方法用生物信息学方法对SPAG4L蛋白进行分析,通过体外转染实验,观察SPAG4L的亚细胞定位;并用免疫荧光技术、免疫共沉淀和双分子荧光互补实验检测SPAG4L是否与KASH结构域蛋白Nesprin-3存在相互作用。结果生物信息学分析结果表明,SPAG4L蛋白具有跨膜结构;亚细胞定位结果发现,SPAG4L蛋白定位于核膜和胞浆;免疫荧光、免疫共沉淀和双分子荧光互补实验结果表明,SPAG4L蛋白与Nesprin-3蛋白质相互作用,形成LINC(linkers of the nucleoskeleton to the cytoskeleton)复合物。结论 SPAG4L与Nesprin-3能够相互作用,形成LINC复合物,对了解SPAG4L蛋白在精子发生中的作用具有重要的意义。
基金supported by the Strategic Priority Research Program of the Chinese Academy of Science(XDB0570000)the CAS Project for Young Scientists in Basic Research(YSBR-009)+3 种基金the National Key Research and Development Program of China(2021YFA1100203,2020YFA0509000)the National Natural Science Foundation of China(31821004,32030029,32293230)Shanghai Municipal Science and Technology Major Project(23HC1401000,22YS1400900)support from the Xplorer Prize and New Cornerstone Science Foundation(NCI202232).
文摘The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels.In this study,we combined the CRISPR-RfxCas13d system with spermlike stem cell-mediated semi-cloning techniques,which enabled the suppressed expression of different RNA species.This approach was employed to interfere with the expression of three types of RNA molecules:Sfmbt2 mRNA,Fendrr lncRNA,and circMan1a2(2,3,4,5,6).The results confirmed the critical roles of these RNAs in embryonic development,as their loss led to observable phenotypes,including embryonic lethality,delayed embryonic development,and embryo resorption.In summary,our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.
文摘在基因编辑领域,以CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)为代表的技术的广泛应用,使得科学家能快速、高效、精确地对靶基因序列进行遗传改造。但是,由于包括人类在内的真核细胞二倍性的特点,要获得纯合编辑的细胞系和模式动物仍需大量的时间和人力成本。类精子干细胞(DKO-AG-haESCs)系统的建立及其与CRISPR-Cas9技术的结合则可以快速地在基因、染色体等水平上进行任意遗传操作并转化为动物个体,实现疾病模拟。本文将对类精子干细胞介导的遗传改造进行总结和讨论。
