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Fig. 1
Transgenic zebrafish lines expressing NLS-tdMCP-eGFP can be used to detect transcripts in vivo. (A) Gateway compatible vectors for generation of NLS-tdMCP-FP and MS2-tagged RNAs. Plasmids used to generate transgenic lines by Tol2-mediated transgenesis are shown. 5′ Entry plasmids (p5E) containing the desired promoter elements can be recombined with NLS-tdMCP-eGFP or NLS-tdMCP-tagRFP entry plasmids (pME) for expression in any cell type or tissue. Similarly, the pME-NLS-tdMCP plasmid can be recombined with any in-frame fluorescent protein (FP) in a 3′ entry plasmid (p3E) to make custom NLS-tdMCP-FPs. RNA localization elements (LE) can be tagged with cherry-24xMBS by recombining the appropriate pME and p3E plasmids. (B) Schematic of the experiment used to validate in vivo MS2-MCP interactions. (C-E) Live imaging of embryos at the sphere stage shows that cytoplasmic puncta are visible only in cells expressing the Cherry reporter. In some cases, as shown in C, the Cherry reporter aggregates, but does not overlap with MCP-GFP cytoplasmic puncta, suggesting that this is independent of the MS2-MCP interaction. The dotted line denotes borders of cells expressing Cherry reporter. Scale bar: 10µm.