Comparative transcriptome analysis of transgenic Drosophila misexpressing novel Notophthalmus viridescens (newt) genes

To obtain a quick glimpse into possible function of five novel Notopthalmus viridescens (newt) genes (and given certain restrains with transgenic newts, such as time) we express these genes using transgenic Drosophila melanogaster. We generated the transgenic flies containing newt candidate genes, and prepared samples for RNA sequencing to evaluate the overall role of candidate genes in tissue development and layout map for future studies. Overall design: We generated five transgenic fly strains each expressing single copy of the five novel Newt genes named as candidate 1 (C1), Candidate 2 (C2), Candidate 3 (C3), Candidate 4 (C4), and Candidate 5 (C5) respectively. Transgenic flies were generated using microinjection-based ?C31 integrase mRNA-mediated method. A cloned candidate gene using pUAST-attB plasmid containing both a transgene and donor sequence (attB) is coinjected along with ?C31 integrase mRNA into attP-containing recipient embryos, resulting in the site-specific insertion of the transgene. Sample (total RNA) for RNA sequencing was collected at 3rd instar larval stage during which major developmental events takes place in Drosophila. Total RNA was extracted, and purified using RNA clean and ConcentratorTM, and high quality RNA samples were used for downstream processes e.g. next-generation RNA sequencing. Of the total 36,099 transcripts in Drosophila, 34,967 transcripts were detected, and 2775 transcripts were significantly regulated by misexpressing these unique gene from newt in Drosophila. Genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched.