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

• DOI: 10.1016/j.molbiopara.2023.111598
• Publication Date: 2023-11-03T19:22:14Z
• Authors: James Budzak; Ione Goodwin; Calvin Tiengwe; Gloria Rudenko
• Keywords: Lac repressor; genomic DNA

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