TRAP-TRAP A SYTEMATIC APPROACH FOR THE DYNAMIC ANALYSIS OF TISSUE-SPECIFIC TRANSCRIPTOMES

THE EMERGENCE OF NEXT-GENERATION SEQUENCING TECHNOLOGIES REPRESENTS A GIANT LEAP TOWARDS THE SYSTEMATIC ANALYSIS OF TRANSCRIPTOMES IN LIVING ORGANISMS. RNA SEQUENCING (RNA-SEQ) 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. AN IMPORTANT LIMITATION IN RNA-SEQ STUDIES COMES FROM THE FACT THAT COMPLEX DEVELOPMENTAL AND PHYSIOLOGICAL PROCESSES DEPEND ON THE ACTIVITY OF HETEROGENEOUS CELL POPULATIONS. THIS RESTRICTS CELL-SPECIFIC TRANSCRIPTOMICS TO DISSECTIBLE TISSUES. ALTERNATIVELY, HOMOGENEOUS CELL SUSPENSIONS CAN BE OBTAINED BY CELL SORTING (FACS), BUT THIS METHODOLOGY DEPENDS ON SEVERE CELL DISSOCIATION PROTOCOLS THAT MAY SIGNIFICANTLY ALTER GENE EXPRESSION. THE TRANSLATING RIBOSOME AFFINITY PURIFICATION (TRAP) APPROACH HAS RECENTLY BEEN DEVELOPED AS A POWERFUL TOOL TO OVERCOME THIS LIMITATION. TRAP METHODOLOGY ALLOWS THE CELL-SPECIFIC RECOVERY OF POLYRIBOSOME-ASSOCIATED RNAS BY GENETIC TAGGING OF RIBOSOMES USING CELL AND TISSUE-SPECIFIC PROMOTERS. IN THE PRESENT PROPOSAL WE PLAN TO COMBINE THE TRAP APPROACH WITH ADAPTED ENHANCER TRAP METHODS IN ZEBRAFISH. THE GOAL IS TO CONDUCT A PILOT SCREEN THAT WILL SYSTEMATICALLY GENERATE ZEBRAFISH TRANSGENIC LINES SUITABLE FOR DYNAMIC AND TISSUE-SPECIFIC TRANSCRIPTOMIC INTERROGATION (TRAP-TRAP APPROACH). THE RESULTING COLLECTION OF LINES MAY CONSTITUTE A UNIQUE RESOURCE FOR A BROAD COMMUNITY WORKING IN DEVELOPMENTAL GENETICS, ORGAN PHYSIOLOGY OR DISEASE MODELS. TO MAKE THESE TRANSGENIC LINES, AS WELL AS THE TECHNOLOGY ITSELF, AVAILABLE TO RESEARCHERS OF THE EXPANDING ZEBRAFISH FIELD, WE PLAN TO TRANSFER THE TRAP-TRAP PROTOCOLS TO THE AQUATIC VERTEBRATES PLATFORM, HOSTED IN OUR RESEARCH CENTRE (CABD). THIS CORE FACILITY WILL HAVE THE CAPACITY TO OFFER TECHNOLOGICAL SUPPORT TO USERS INTERESTED IN THE TISSUE-SPECIFIC TRANSCRIPTOMIC ANALYSES PROVIDED BY THE TRAP-TRAP APPROACH.