Fig. 2.
Fluorescent labeling methods. (A) A construct designed for in vitro transcription of the gene of interest from a bacteriophage promoter (T7 in this example), with the coding region shown in gray and the 3′UTR containing the RNA localization signal in red. Transcription of this construct by T7 polymerase in the presence of a fluorophore-coupled nucleotide produces fluorescently labeled RNA for injection into cultured egg chambers. RNA can be injected directly into the oocyte as illustrated or into the nurse cells. (B) In vivo labeling of endogenous mRNA by the MS2 system. This strategy requires two components: a transgene (transgene 1) that encodes the target RNA with an insertion of tandem copies of the stem-loop binding site for the bacteriophage MS2 coat protein (MCP), shown here in the 3′ UTR, usually under the control of its own promoter (Px); and a transgene (transgene 2) that encodes a fluorescent protein fused to MCP (GFP is shown here) under the control of a maternally active promoter (Pmat). Transgenic fly lines for each component are crossed together to generate females that express both the tagged RNA and the MCP-GFP protein in their ovarian nurse cells. When the two transgenes are thus coexpressed, the binding of MCP to its recognition motif labels the RNA with GFP. The nuclear localization signal (NLS) in the MCP-GFP fusion protein retains excess unbound protein in the nucleus, reducing cytoplasmic background. Fluorescently labeled mRNA enters the oocyte from the nurse cells (not shown). (C) Transgenic expression of GFP-tagged localization factors. A transgene encoding a localization factor fused to a fluorescent protein (e.g. GFP) under the control of its own or a maternally active promoter. Expression of the transgene in the nurse cells of transgenic females will result in production of the fusion protein in the nurse cells. Colocalization of the fusion protein with the target RNA could occur in the nurse cells or oocyte (not shown), through direct RNA-protein interaction (as shown) or through their co-assembly into a larger RNP.