Sirna-mediated Rna Interference In Zebrafish And Dec1/sharp2/stral3 Gene Cloning And Functional Study

RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) and causes degradation of its homologous mRNAs. RNAi has been observed in a wide range of eukaryotes, including fungi, plants and animals. In vertebrates, long dsRNA activates the interferon response and yield non-specific degradation of mRNA. In contrast, small interference RNA (siRNA) duplexes with the length of 21 to 23 nucleotides trigger specific gene silencing and thus be widely used for gene functional studies. The use of siRNA for gene silencing in zebrafish has rarely been reported. In this thesis, I studied U6 promoter-driven siRNA mediated RNA interference in zebrafish. Four characterized genes Myf5, Dlg3, Mitfa/Nacre, Mitfb, and a reporter gene were selected as targeted genes. Two to four target siRNAs were synthesized with U6 promoter for each of them, respectively. Constructs were introduced into early zebrafish embryos through microinjection, followed by in situ hybridization and embryonic development observation to explore whether U6 promoter-driven siRNAs could efficiently suppress specific gene expression in zebrafish embryo. I showed that, 1) U6 promoter-driven siRNAs could partially suppress endogenous gene expression; 2.) siGFP showed no obvious repression to GFP expression; 3.) The siRNA efficiency varies with different targeted positions. Potential siRNA triggered non-specific suppression is under further study.DEC1 (also known as Sharp2 or Stra13) is a basic helix-loop-helix (bHLH) transcription factor that plays key roles in embryonic development, cell differentiation, proliferation and apoptosis in mammal. DEC1 is a regulator of the mammalian molecular clock, and form a fifth clock-gene family. In this report, I describe the cloning of a zebrafish homologue of mammalian DEC1 and bioinformatic analysis of its expression during early development. RT-PCR analysis detected the expression of zebrafish DEC1 mRNA in adult eyes under LD cycles. Furthermore, we explored the developmental function of DEC1 with morpholino antisense oligonuleotide (MO) and overexpression methods. Here I report: 1) The full-length cDNA of zebrafish DEC1 is of 2743 bp which encodes a protein of 403 amino acids. Zebrafish DEC1 is closely related to tetraodon, human and mouse homologues, displaying 64%, 60%, 59%, 46% and 39% identity to tetraodon (CAAE01014528.1), mouse musculus (NM₀11498.2), human (NM₀03670.1), Xenopus (BC073563.1) and Canis (AY204568.1), respectively. Zebrafish DEC1 contains highly conserved bHLH domain and belongs to the DEC1 family of basic helix-loop-helix transcription factors. 2) DEC1 transcripts are widely and strongly expressed before early segmentation period (4-somites stage); it also expressed in eyes, somites, blood islands, and pineal later on; By Pec-fin stage (60 hpf), the expression signal in somites and other tissues declined while strong expression in pineal and RPE continues. By Protruding-mouth stage (72hpf), DEC1 was restricted in pineal and RPE. DEC1 is expressed in adult zebrafish eyes rythemly. 3.) Overexpression and MO-knockdown of DEC1 result in seriously phenotypic abnormalities, as the short body, abnormal and suspended tail bud and abnormal heart and vessels; some embryos were even growth arrested. We speculate that 1) DEC1 is probably involved in zebrafish circadian clock system, and thus could be considered as a a clock gene candidate; 2) DEC1 may play critical roles in embryonic cell differentiation in several tissues including somites and early hematopoisis organs in zebrafish.