On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.

Autor:	Gemperle J; Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom., Harrison TS; Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom., Flett C; Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom., Adamson AD; Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom., Caswell PT; Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.
Jazyk:	angličtina
Zdroj:	ELife [Elife] 2022 Jun 16; Vol. 11. Date of Electronic Publication: 2022 Jun 16.
DOI:	10.7554/eLife.76651
Abstrakt:	CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon ( D oxycycline-mediated endogenous gene Ex pression Con trol), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines. Competing Interests: JG, TH, CF, AA, PC No competing interests declared (© 2022, Gemperle et al.)
Databáze:	MEDLINE
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