Background
Next-generation sequencing technologies have contributed enormously to the systematic analysis of transcriptomes in living organisms. The RNA-seq technique has been established as the ultimate tool to explore the expression profile of cells and tissues, providing a precise, unbiased, and genome-wide measurement of transcript levels. However, an important limitation of this methodology arises when gene expression needs to be analyzed in small tissues or in specific cell types within heterogeneus populations. Mechanical cell separation involves great technical difficulty and it increases the risk of mixing the expression profiles of neighboring cells.
The TRAP methodology emerged to overcome this important limitation (Doyle et al., 2008; Heiman et al., 2008; Tryon et al., 2012). This technique allows the direct extraction of transcriptomes from specific cells thanks to marking their ribosomes with the fluorescent protein GFP. Then, through immunoprecipitation it is possible to isolate the mRNA attached to the marked ribosomes. Nevertheless, the availability of these specific promoters, and the need to create different lines for each cell type, restrict the applicability of TRAP.
THE TRAP-TRAP TECHNOLOGY
The trap-TRAP approach eliminates the constraints of TRAP methodology. Using this technology we have been able to systematically and succesfully generate diverse TRAP lines in a quick, easy and unbiased manner in zebrafish. These lines are appropriate for tissue-specific transcriptomic interrogation in different developmental stages or different physiological situations. They constitute a unique transcriptomic resource for a broad community working in developmental genetics, organ physiology or disease models among others.
This website displays information about the trap-TRAP collection lines, which are available to the scientific community upon request.
Methods
The TRAP (Translating Ribosome Affinity Purification) methodology consists in tagging the ribosomal large subunit protein L10a (rpl10a) with the fluorescent protein GFP. The expression of this fusion protein can be controlled by a specific promoter which is able to direct its expression to specific tissues. Therefore, the cells where this promoter is active in will contain tagged ribosomes. Using inmunoprecipitation techniques it is possible to isolate the tagged ribosomes using antibodies against the fusion protein. The isolated ribosomes will have attached mRNA which can be purified getting the mRNA from a specific tissue. After this, through RNAseq the transcriptomic (translatomic) status of the chosen cells can be assessed.
The trap-TRAP technology is based in the combination of the TRAP methodology with the enhancer trap analysis method (Fig. 1). Enhancer trap screens are based in the random insertion in the genome of a reporter gen, such as LacZ, GFP or Gal4, downstream of a minimal promoter. When this construct gets inserted close to an enhancer, its expression is activated.
Fig. 1. Schematic representation of the enhancer trap configuration. Adapted from Trinh and Fraser, 2013
In the trap-TRAP method, the reporter gene is replaced by the fusion gene eGFP-rpl10a. This fusion gene is set under the control of the Gata2p minimal promoter which can be activated by nearby regulatory elements (e.g. enhancers) once integrated in the genome. A plasmid containing this genetic cassete was created towards the trap-TRAP transgenic zebrafish lines generation (Fig. 2)
