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.

通过显微镜观察基因组位点对于理解核组织至关重要，特别是在单细胞水平上。研究基因组位点定位的一项强大技术是通过 Lac Operator-Lac Repressor (LacO-LacI) 系统，其中可以通过与荧光标签融合的 LacI 蛋白的表达来可视化引入特定基因组位点的 LacO 重复序列。20 多年前首次在布氏锥虫中使用，我们现在通过小于 2 kb 的短稳定 LacO 重复序列与组成型表达的 mNeongreen::LacI 融合蛋白配对来优化该系统，以促进基因组位点的可视化。我们证明了该系统与超分辨率显微镜的兼容性，并提出其适合与诱导性 RNAi 或蛋白质过度表达进行多重分析，这将允许在基因表达扰动后分析核组织。