Fluorescent Tagging of Endogenous FOXO for Live Imaging and Pull-Down Assays.

Abstract

Proteins are the major and most diverse biomolecules, directing all activities of a cell. For this reason, visualizing protein expression, localization, and dynamics is fundamental in biology. In most cases, protein visualization relies on the overexpression of fluorescently tagged proteins which may not recapitulate endogenous expression pattern and dynamics. Henceforth, tagging proteins in the endogenous locus is the most accurate way to recapitulate physiological gene expression. However, this method is not widely implemented for the FOXO gene family due to its technical inefficiency and difficulty. Here we describe the methodology followed to generate a knock-in reporter line for the Foxo1a transcription factor for the zebrafish model system. We describe insertion of an EGFP-polyA cassette in frame at the C-terminal of Foxo1a, generating a fusion protein. Foxo1a has been involved in the regulation of metabolism, stress response, longevity, and cell differentiation, and its functions are conserved from invertebrates to vertebrates. Using in vivo confocal live microscopy at early developmental stages, we validated the expression of Foxo1a in the cardiovascular network, central nervous system, olfactory epithelium, spinal cord, retina, skeletal muscle, and myocardium. This knock-in line opens the way for imaging studies aiming to characterize the expression and localization (cytoplasmic or nuclear) of this transcription factor in a tissue- and context-specific manner, as well as the dynamics of stress adaptation at a whole organism level. Moreover, the knock-in line can be used in combination with other modern techniques such as Cut&Run to determine the transcriptional targets of Foxo1a, with a GFP-directed proteomic to identify interacting partners, many of which remain largely unknown.