Tracing endogenous proteins in living cells through electrotransfer of mRNA encoding chromobodies.
Fiche publication
Date publication
février 2024
Journal
Biotechnology journal
Auteurs
Membres identifiés du Cancéropôle Est :
Pr CHATTON Bruno, Dr MASSON Murielle, Dr ZUBER Guy, Dr DONZEAU Mariel, Dr RICHERT Ludovic
Tous les auteurs :
Juncker T, Richert L, Masson M, Zuber G, Chatton B, Donzeau M
Lien Pubmed
Résumé
Chromobodies made of nanobodies fused to fluorescent proteins are powerful tools for targeting and tracing intracellular proteins in living cells. Typically, this is achieved by transfecting plasmids encoding the chromobodies. However, an excess of unbound chromobody relative to the endogenous antigen can result in high background fluorescence in live cell imaging. Here, we overcome this problem by using mRNA encoding chromobodies. Our approach allows one to precisely control the amount of chromobody expressed inside the cell by adjusting the amount of transfected mRNA. To challenge our method, we evaluate three chromobodies targeting intracellular proteins of different abundance and cellular localization, namely lamin A/C, Dnmt1 and actin. We demonstrate that the expression of chromobodies in living cells by transfection of tuned amounts of the corresponding mRNAs allows the accurate tracking of their cellular targets by time-lapse fluorescence microscopy.
Mots clés
chromobody, fluorescence microscopy, live cell imaging, mRNA, nanobody, time-lapse
Référence
Biotechnol J. 2024 02;19(2):e2300548