Analyzing the Translatome of Lymphatic and Venous Endothelial Cells In Vivo via Translating Ribosome Affinity Purification (TRAP).

Zebrafish are a powerful model for investigating vascular and lymphatic biology due to their genetic tractability and optical transparency. While translating ribosome affinity purification (TRAP) has been widely applied in other systems, its application in zebrafish has remained limited. Here, we present an optimized TRAP protocol for isolating ribosome-associated mRNAs from endothelial cells in vivo, without the need for cell dissociation or sorting. Using a novel transgenic zebrafish line, which expresses HA-tagged Rpl10a under the mrc1a promoter, we enriched actively translating endothelial transcripts. Differential expression analysis revealed robust upregulation of vascular and lymphatic genes including flt4, kdrl, and lyve1b. This approach captures the endothelial cell translatome with high specificity and offers a robust platform for investigating the molecular mechanisms of endothelial biology under genetic, environmental, or toxicological perturbations. Key features • Permits in vivo isolation of the endothelial ribosome without cell sorting. • Optimized TRAP protocol to analyze lymphatic and venous endothelial gene expression in zebrafish. • Utilizes a stable transgenic zebrafish line. • Compatible with real-time quantitative PCR (qPCR) and next-generation sequencing (RNA-seq).