MicroRNA Protocols

Shi-Lung Lin, Shin-Ju E. Chang, Shao-Yao Ying, Lin, Shi-Lung, Chang, Shin-Ju E., Ying, Shao-Yao

Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude for a human body for etiological and pathological research of diseases. All pharmaceutically developed drugs must be proven to be safe and effective in animals before approval by the Food and Drug Administration to be used in clinical trials. To this end, the transgenic animal models of diseases serve as the front line of drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA) research. miRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts (mRNAs) that contain either complete or partial complementarity to the miRNA, are useful for the design of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis spp.), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse, and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerases type II (Pol II), coexpressed with the encoding gene transcript, and excised out of the encoding gene transcript by natural RNA splicing and processing mechanisms. In conjunction with retroviral transfection systems, the designed hairpin-like pre-miRNA construct was further tested to insert into the intron regions of a cellular gene for tissue-specific expression regulated by the gene promoter. Because the retroviral vectors were randomly integrated into the genome of its host cell, the most effective transgenic animal can be selected and propagated to be a stable transgenic line for future research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA-expressing system described previously, which has been proven to be useful for both miRNA research and in vivo evaluation of miRNA-associated target gene functions.