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Maliheh Davari, Hamid Ahmadieh, Zahra-Soheila Soheili, Shahram Samiei, Ehsan Ranaei, Mozhgan Rezaei Kanavi; Construction of Recombinant AAV-based Vectors bearing miR-183 Cluster Genes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3465.
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miR-183 cluster as a sensory organ-enriched miRNA cluster has been recognized to be an important class of retina-specific miRNAs. Members of miR-183 cluster (miR-183, miR-96 and miR-182) play a role in development and survival of the neuro-retina particularly photoreceptors. This study aimed to construct recombinant AAV-based vectors containing miR-183 cluster genes.
AAV Helper-Free System was purchased from Agilent Technologies. miR-183, miR-96 and miR-182 complete sequences were amplified from human genome through PCR using specific linker primer pairs with suitable restriction sites at their 5'-ends. Double-digested PCR products corresponding to each miRNA sequence, as well as IRES-GFP fragment providing green fluorescence gene as a reporter gene, were cloned into pAAV-MCS vector. To check the accuracy of cloning of all three miRNA genes into pAAV-MCS vectors, the resultant constructs were subjected to digestion and sequencing experiments.
Desired recombinant pAAV-MCS vector bearing all three miR-183 cluster genes simultaneously followed by GFP gene was constructed. On the other hand, recombinant pAAV-MCS vectors bearing each of three miRNAs were individually constructed.
Given critical roles of miR-183 cluster in neuro-retina maintenance and survival and high efficiency of AAV-based vectors to provide high and stable gene expression levels in various tissues; the resultant constructs would be applied to study the over-expression effects of miR-183 cluster genes on different parts of the retina.
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