Study Of Short Interfering RNA (siRNA) Produced In Transcription System Of Zebrafish Embryo

RNA interference (RNAi) is the phenomenon of post-transcriptional gene silencing (PTGS) in which dsRNA specifically blocks the expression of its homologous gene. This process is found in C. elegans in 1998 and is the mechanism existed in most eukaryotes responsible for resistance to virus intrusion, inhibition of transponson activities and gene expression regulation. RNAi has been widely applied in many organisms such as plants, lower animals and higher vertebrates. The study in lower animals focuses on the mechanism of RNAi, and in higher vertebrates such as mammals, it has been applied to study of gene function, signal pathway and gene therapy.Zebrafish (Danio rerio) is a kind of teleost which belongs to Danio genus, Cyprinidae family, Actinopterygii class. Zebrafish is a widely used model animal for genetic studies including vertebrate development, genetics, pharmacology, toxicology and environmental protection owing to its advantages as a research tool including a short life cycle, high production of eggs, and in vitro fertilization and in vitro embryogenesis.However, as a model organism receiving more and more attention, the study of gene function in zebrafish has not developed with convenient technology as that in the lower animals and mouse. The appearance of RNAi as a tool to study gene function attracted many researchers to investigate the possibility of RNAi in analysis of gene function. Whereas, so far, reports on the study of RNAi in zebrafish have not reached the consistent conclusions, but even controversial conclusions. Thus, it remains a question that whether RNAi works in zebrafish as a tool to study gene function. Further work is required to discuss the application of RNAi in zebrafish. This project aims to establish the application platform for RNAi in zebrafish by employing the zebrafish H1 and U6 promoters and T7 transcription system to provide the foundation for gene function and signal pathway study in zebrafish. To achieve these goals, the several aspects was carried out as follows.1. Cloning and transcription analysis of zebrafish H1 and U6 promoters. Zebrafish H1 and U6 promoters were firstly identified from zebrafish genome database by BLAST with zebrafish H1 RNA and U6 snRNA gene. By comparison with H1 and U6 promoters of other organisms, three conserved regions required for promoter activity were found: DSE, PSE and TATA. These two promoters were cloned by PCR and ligated with a DNA sequence in similar length with H1 RNA and U6 RNA to form transcription vectors. The transcription activity was analyzed by injecting these vectors into zebrafish embryos and the results showed that the zebrafish H1 and U6 promoters could drive the transcription of small RNA and the transcription of mRNA as well.2. The application of shRNA vectors driven by zebrafish H1 and U6 promoters in zebrafish. To analyze the function of zebrafish H1 and U6 promoters in RNAi, short hairpin RNA (shRNA) vectors driven by H1 and U6 promoters targeting No tail (ntl) were constructed and were firstly co-transfected into common carp epithelioma cells (EPC) with pGL3-control-NTL vector. The results showed that H1 and U6 promoter could work in fish cells and block the expression of target gene specifically. Further shRNA vectors targeting GFP and NTL were injected into zebrafish embryos and the results revealed that the expression of the target genes was down-regulated when analyzed by fluorescence real-time RT-PCR and whole mount in situ hybridization. 3. The construction and confirmation of vectors used for application of T7 system in zebrafish RNAi: A short-hairpin RNA (shRNA) expression system included T7 RNA polymerase expression vector and shRNA expression vector has been developed based on T7 RNA polymerase (T7RP) directed transcription machinery. Firstly the germline transmitted transgenic individuals were screened out in transgenic pCMVT7R zebrafish. By using bmp2b construct driven by T7 promoter, we validated the transcription activity of T7 RNA polymerase in pCMVT7 transgenic F3 embryos. In addition, the vectors pT7shRNA were injected into the F3 embryos and in vivo transcription of shRNA was detected by RT-PCR analysis. These results confirmed the feasibility of T7 system in zebrafish and provided a foundation for RNAi analysis.4. The application of T7 system in zebrafish RNAi. The inhibition efficiency was analyzed after pT7shRNA was injected into transgenic zebrafish embryos stably expressing T7 RNA polymerase. After pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp mRNA relative expression level showed a decrease of 68% than the control analyzed with fluorescence real-time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in a decreased expression level of 40% lower than the control when the injection dose was as high as 2ug/ul. Injection of pT7 shNTL vector in zebrafish embryos expressing T7 RNA polymerase led to the partial absence of ntl transcripts in 30% of the injected embryos detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down, suggesting a potential role for its application in RNAi studies in zebrafish embryos.In summary, this project firstly described the cloning, transcription activity analysis and RNAi application of zebrafish H1 and U6 promoters. By ways of experiments in cultured fish cells and zebrafish embryos, shRNA vectors driven by zebrafish H1 and U6 promoters could knock down the transcription levels of foreign and endogenous genes. This study also applied bacteriophage T7 RNA polymerase/T7 promoter system in RNAi study of zebrafish embryos and the results confirmed that this system could work well in zebrafish embryos. Furthermore, shRNA vector driven by T7 promoter inhibited effectively the target genes expression in transgenic zebrafish embryos which expressed stably T7 RNA polymerase. This study provided a good foundation for further application of RNAi in zebrafish and a wide profoundness for study gene function in zebrafish by ways of RNAi.