| Summary: | 碩士 === 國立臺灣大學 === 畜產學研究所 === 93 === It is well recoginized that the internal ribosome entry site (IRES) existed within the host cells is essentially required for translation in some species of viral RNA infected and this is particularly true in those of poliovirus. Moreover, there was a group of small RNA found within the yeast and these small RNA were characterized as an inhibitor RNA (IRNA) by showing capability in a selective-blocking the IRES-mediated translation of the infected viral RNA without resulting in interference of the cap-dependent translation in the host cells. Studies of using single-strand- and double-strand specific nucleases have predicted the secondary structure of IRNA consisting two loops, a 7-base long stem, and a large bugle region. Based on the fact that IRNA inhibits viral IRES-mediated translation can also be achieved by sequestering noncanonical transacting factors and canonical factors, both essential for IRES-mediated translation, attempts of the present studies were made to investigate the feasibility of using IRNA as a strategy for prevention the diseases suffer from specific viral infections.
To meet the purpose described above, a fragment length in 71 bp of the IRNA sequences driving by a H1 promoter was constructed into the pSuper-Neo+GFP vector to allow the transgene equipped with both the Neomycin ORF and the EGFP ORF. The transgene, named as pSuper-Neo+GFP-IRNA, after construction was first subjected to transfection into Vero cells and the efficiency of IRNA against to the IRES-mediated translation was evaluated after the transient transfected Vero cells had been challenged with enterovirus 71. Evidences from cell morphology examination appeared that majority of the control Vero cells showing remarkable cytopathic effect after they had been challenged enterovirus 71 when comparisons were made to those of transient transfected Vero cells, indiciating that IRNA did express their inhibitory effect on the IRES-mediated viral translation and subsequently resulting in inhibition of the enterovirus replication. Therefore, further studies were made to use the transgenic mice harboring the IRNA-transgene as an animal model for elucidation the function of IRNA in IRES-mediated viral translation in vivo and the generation of transgenic mice was conducted by microinjecting the IRNA transgene into pronucleus of newly fertilized eggs. Of these studies, a total 866 mouse embryos after gene injection were reimplanted into the recipients and 83 newborn mice have so far been obtained. Further verification of the transgene integrated within their genomic DNA and also the ability of the potential transgenic mice possess resistance of those disease suffered from specific viral infections are now in progression.
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