Imaging of genomic loci in Trypanosoma brucei using an optimised LacO-LacI system

IF 1.4 4区医学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Molecular and biochemical parasitology Pub Date : 2023-11-03 DOI:10.1016/j.molbiopara.2023.111598

James Budzak , Ione Goodwin, Calvin Tiengwe, Gloria Rudenko

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引用次数: 0

Abstract

Visualisation of genomic loci by microscopy is essential for understanding nuclear organisation, particularly at the single cell level. One powerful technique for studying the positioning of genomic loci is through the Lac Operator-Lac Repressor (LacO-LacI) system, in which LacO repeats introduced into a specific genomic locus can be visualised through expression of a LacI-protein fused to a fluorescent tag. First utilised in Trypanosoma brucei over 20 years ago, we have now optimised this system with short, stabilised LacO repeats of less than 2 kb paired with a constitutively expressed mNeongreen::LacI fusion protein to facilitate visualisation of genomic loci. We demonstrate the compatibility of this system with super-resolution microscopy and propose its suitability for multiplexing with inducible RNAi or protein over expression which will allow analysis of nuclear organisation after perturbation of gene expression.

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使用优化的LacO-LacI系统对布鲁氏锥虫基因组基因座进行成像。

通过显微镜观察基因组基因座对于理解核组织至关重要，尤其是在单细胞水平上。研究基因组基因座定位的一种强大技术是通过Lac-Operator Lac-Repressor（LacO-LacI）系统，其中引入特定基因组基因座的LacO重复序列可以通过表达与荧光标签融合的LacI蛋白来可视化。20多年前，我们首次在布鲁氏锥虫中使用，现在我们已经用小于2kb的稳定的短LacO重复序列与组成型表达的mNeongreen:：LacI融合蛋白对该系统进行了优化，以促进基因组基因座的可视化。我们证明了该系统与超分辨率显微镜的兼容性，并提出其适用于与诱导型RNAi或蛋白质过表达进行多路复用，这将允许分析基因表达扰动后的核组织。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular and biochemical parasitology 医学-寄生虫学

CiteScore

2.90

自引率

0.00%

发文量

审稿时长

63 days

期刊介绍： The journal provides a medium for rapid publication of investigations of the molecular biology and biochemistry of parasitic protozoa and helminths and their interactions with both the definitive and intermediate host. The main subject areas covered are: • the structure, biosynthesis, degradation, properties and function of DNA, RNA, proteins, lipids, carbohydrates and small molecular-weight substances • intermediary metabolism and bioenergetics • drug target characterization and the mode of action of antiparasitic drugs • molecular and biochemical aspects of membrane structure and function • host-parasite relationships that focus on the parasite, particularly as related to specific parasite molecules. • analysis of genes and genome structure, function and expression • analysis of variation in parasite populations relevant to genetic exchange, pathogenesis, drug and vaccine target characterization, and drug resistance. • parasite protein trafficking, organelle biogenesis, and cellular structure especially with reference to the roles of specific molecules • parasite programmed cell death, development, and cell division at the molecular level.