在黑腹果蝇 OSC 细胞培养中诱导性敲除重要基因的方法

IF 1.5 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Molecular Biology Pub Date : 2024-04-09 DOI:10.1134/s0026893324020110

S. V. Marfina, E. A. Mikhaleva, N. V. Akulenko, S. S. Ryazansky

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引用次数: 0

摘要

摘要提出了一种基于 RNA 干扰的方法，以实现对细胞活力至关重要的基因的诱导性敲除。在该方法中，通过 CRIPSR/Cas9 技术将铜离子依赖性诱导型金属硫蛋白启动子控制 siRNA 前体表达的基因盒插入基因组预整合转基因中。这种内源 siRNA 可用于基因敲除细胞培养物，而传统的外源 siRNA 转染方法对这种细胞培养物无效。该方法在果蝇卵巢体细胞培养（OSC）中对两个卵子发生所必需的基因进行了高效验证：Cul3编码多蛋白泛素连接酶复合物的一个成分，在蛋白稳态中具有多种功能；cut编码一个转录因子，调节卵巢滤泡细胞的分化。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

Method of Inducible Knockdown of Essential Genes in OSC Cell Culture of Drosophila melanogaster

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Method of Inducible Knockdown of Essential Genes in OSC Cell Culture of Drosophila melanogaster

Abstract

An RNA interference-based method was proposed to achieve an inducible knockdown of genes essential for cell viability. In the method, a genetic cassette in which a copper ion-dependent inducible metallothionein promoter controls expression of a siRNA precursor is inserted into a genomic pre-integrated transgene by CRIPSR/Cas9 technology. The endogenous siRNA source allows the gene knockdown in cell cultures that are refractory to conventional transfection with exogenous siRNA. The efficiency of the method was demonstrated in Drosophila ovarian somatic cell culture (OSC) for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating differentiation of ovarian follicular cells.

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来源期刊

Molecular Biology 生物-生化与分子生物学

CiteScore

1.30

自引率

8.30%

发文量

审稿时长

3 months

期刊介绍： Molecular Biology is an international peer reviewed journal that covers a wide scope of problems in molecular, cell and computational biology including genomics, proteomics, bioinformatics, molecular virology and immunology, molecular development biology, molecular evolution and related areals. Molecular Biology publishes reviews, experimental and theoretical works. Every year, the journal publishes special issues devoted to most rapidly developing branches of physical-chemical biology and to the most outstanding scientists.