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The knockdown efficiency of telomere associated genes with specific methodology in a zebrafish cell line
Biochimie ( IF 3.9 ) Pub Date : 2021-06-30 , DOI: 10.1016/j.biochi.2021.06.013
Xuefei Hu 1 , Shuaiyun Gao 1 , Peng Wang 1 , Yulin Zhou 2 , Kehua Chen 1 , Qiaowen Chen 1 , Bo Wang 1 , Weiguo Hu 3 , Peng Cheng 1 , Rita Eid 4 , Marie-Josèph Giraud-Panis 4 , Lei Wang 3 , Eric Gilson 5 , Jing Ye 1 , Yiming Lu 1
Affiliation  

Zebrafish is broadly used as a model organism in gene loss-of-function studies in vivo, but its employment in vitro is greatly limited by the lack of efficient gene knockdown approaches in zebrafish cell lines such as ZF4. In this article, we attempted to induce silencing of telomere associated genes in ZF4 by applying the frequently-used siRNA transfection technology and a novel moiety-linked morpholino (vivo-MO). By proceeding with integrated optimization of siRNAs transfection and vivo-MOs treatment, we compared five transfection reagents and vivo-MOs simultaneously to evaluate the efficiency of terfa silencing in ZF4. 48 h after siRNAs transfection, Lipofectamine™ 3000 and X-tremeGENE™ HP leaded to knockdown in 35% and 43% of terfa transcription, respectively, while vivo-MO-terfa modulated 58% down-expression of zfTRF2 in contrast to vivo-MO-ctrl 72 h after treatment. Further siRNAs transfection targeting telomere associated genes by X-tremeGENE™ HP showed silencing in 40–68% of these genes without significant cytotoxicity and off-target effect. Our results confirmed the feasibility of gene loss-of-function studies in a zebrafish cell line, offered a systematic optimizing strategy to employ gene silencing experiments, and presented Lipofectamine™ 3000, X-tremeGENE™ HP and vivo-morpholinos as candidate gene silencing approaches for zebrafish in vitro gene loss-of-function studies. Successfully knockdown of shelterin genes further opened a new field for telomeric study in zebrafish.



中文翻译:

斑马鱼细胞系中端粒相关基因的敲除效率与特定方法

斑马鱼在体内基因功能丧失研究中被广泛用作模型生物,但由于在 ZF4 等斑马鱼细胞系中缺乏有效的基因敲除方法,它在体外的应用受到很大限制。在本文中,我们尝试通过应用常用的 siRNA 转染技术和一种新型的部分连接的吗啉代 (vivo-MO) 来诱导 ZF4 中端粒相关基因的沉默。通过对 siRNAs 转染和 vivo-MOs 处理进行综合优化,我们同时比较了五种转染试剂和 vivo-MOs,以评估terfa沉默在 ZF4 中的效率。siRNA 转染 48 小时后,Lipofectamine™ 3000 和 X-tremeGENE™ HP 导致 35% 和 43% 的terfa被敲低分别转录,而处理后 72 小时,与 vivo-MO- ctrl相比,vivo-MO- terfa调节了 58% 的 zfTRF2 下调。通过 X-tremeGENE™ HP 进一步转染靶向端粒相关基因的 siRNA 显示 40-68% 的这些基因沉默,没有显着的细胞毒性和脱靶效应。我们的结果证实了在斑马鱼细胞系中进行基因功能丧失研究的可行性,提供了采用基因沉默实验的系统优化策略,并提出 Lipofectamine™ 3000、X-tremeGENE™ HP 和 vivo-morpholinos 作为候选基因沉默方法用于斑马鱼体外基因功能丧失研究。成功敲除shelterin基因进一步开辟了斑马鱼端粒研究的新领域。

更新日期:2021-07-08
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