Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels, Belgium.
Methods Mol Biol. 2025;2871:145-153. doi: 10.1007/978-1-0716-4217-7_13.
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.
蛋白质是主要的、最多样化的生物分子，指导着细胞的所有活动。因此，可视化蛋白质的表达、定位和动态是生物学的基础。在大多数情况下，蛋白质的可视化依赖于荧光标记蛋白的过表达，而过表达的蛋白可能无法重现内源性的表达模式和动态。因此，在内源性基因座上标记蛋白质是重现生理基因表达的最准确方法。然而，由于其技术效率低和难度大，这种方法在 FOXO 基因家族中并未得到广泛应用。在这里，我们描述了为斑马鱼模型系统生成 Foxo1a 转录因子敲入报告系的方法。我们描述了在 Foxo1a 的 C 末端插入 EGFP-polyA 盒，生成融合蛋白。Foxo1a 参与代谢、应激反应、长寿和细胞分化的调节，其功能从无脊椎动物到脊椎动物都是保守的。通过在早期发育阶段进行体内共聚焦活显微镜检查，我们验证了 Foxo1a 在心血管网络、中枢神经系统、嗅上皮、脊髓、视网膜、骨骼肌和心肌中的表达。这种敲入系为旨在以组织和上下文特异性方式表征这种转录因子的表达和定位（细胞质或核）以及在整个生物体水平上适应应激的动态的成像研究开辟了道路。此外，敲入系可以与其他现代技术（如 Cut&Run）结合使用，以确定 Foxo1a 的转录靶标，用 GFP 指导的蛋白质组学来鉴定相互作用的伙伴，其中许多仍然知之甚少。
