The Research On Toxicities In ShRNA-transgenic Swine

RNA interference (RNAi) is a post-transcriptional phenomenon caused by double-strandedRNA. With further research on mechanism of RNAi, it was found that shRNA and miRNA sharesimilar processing and transport pathways including Dicer, silencing complex (RISC) and otherenzymes. Then subsequent studies confirmed that the application of RNAi also encountered somechallenges, such as off-target effects, non-specific immune response reaction, saturation ofmiRNA pathway and so on, which hinder the research on shRNA-transgenic animals. Cellreprogramming that occurs in the process of somatic cell nuclear transfer (SCNT) may convertexogenous shRNA cassettes into endogenous miRNAs. Whether in vivo toxicities induced byintravenously injected exogenous siRNA/shRNA would occur in shRNA-transgenic animalsremains controversial. At present, many measures incuding chemical modification andoptimization of sequence have been adapted to avoid non-specific immune response, off-targeteffects. However, saturation of miRNA pathway has not been resolved. Thus, how to screen singlecopy, low expression levels, efficientive shRNA has become a focus recently.In our study, a low survival rate and early lethality were observed in shRNA-transgenic pigscompared with other transgenic pigs when we attempted to produce shRNA-transgenic pigs withanti-classical swine fever virus (CSFV) capacity. To assess the effect on transgenic pigs inducedby shRNA, we generated two types of shRNA-transgenic pigs (shRNA-NS3-2, shRNA-NS5).Samples obtained from the clonal pigs, donor cells, recipients were analyzed by using ShortTandem Repeat (STR), PCR and copy number detection method and results confirmed thatshRNA had been intergrated into genome of pigs. In order to make a preliminary assessment ofantivirus ability of shRNA, cells were isolated from transgenic piglets and results of Indirectimmunofluorescence assay (IFA) indicated shRNA can inhibit CSFV replication significantly.Results of RT-PCR and Western Blotting indicated that shRNA induce abnormal expression levelsof endogenous microRNAs and their processing enzymes in livers of transgenic pigs. These resultsshowed that shRNA cause disruption of endogenous miRNA pathway in transgenic pigs, whichmay lead to the early lethality of shRNA-transgenic pigs. In order to minimize toxicities induced by RNAi and identify potent single copy, lowexpression levels, efficientive shRNA, we developed a fluorescent reporter assay that enablesidentification of potent shRNAs. We constructed a reporter vector harboring an shRNA expressedunder the control of a Tet-responsive element (TREtight) and its cognate target sequence (Sensor) inthe3’ UTR of a constitutively expressed fluorescent reporter. We combined it with retroviraltransfection and Fluorescence Activated Cell Sorter (FACS) that functionally monitors theinteraction of shRNAs with their specific target sites. We constructed two pSENSOR vectors tovalidate the potency of shRNAs against CSFV based on two pre-existing anti-CSFV siRNA-N2,siRNA-NS5B. Our results showed that shRNA-N2has a intermediate potency and shRNA-NS5Binhibits its target site weakly and confirmed different design principles between siRNA andshRNA. In our study, the new method greatly improves the sensitivity of identifying shRNAs,provides the guarantee for research of shRNA-transgenic animal preparation.This study reported early lethality of shRNA-transgenic animals caused by disruption ofendogenous miRNA pathway and developed a highly sensitive fluorescent reporter assay thatenables identification of potent shRNAs, providing the guarantee for research ofshRNA-transgenic animal preparation.