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 medicines must be proven to be safe and effective in animals before approval by the Food and Drug Administration (FDA) 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)-related 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 be 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 the 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 transcripts, and excised out of the encoding gene transcripts by intracellular RNA splicing and processing mechanisms. In conjunction with retroviral transfection, the designed hairpin-like pre-miRNA construct has also been placed in the intron regions of a cellular gene for tissue-specific expression, specifically regulated by the gene promoter of interest. Because the retroviral vectors are integrated into the genome of its host cells, we can select and propagate the most effective transgenic animals to form a stable model line for further research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA expression system reported previously, which has been proven to be useful for studying miRNA function as well as the related gene regulation in vivo.