Lin Shi-Lung, Chang Shin-Ju E, Ying Shao-Yao
Division of Regenerative Medicine, WJWU and LYNN Institute for Stem Cell Research, Santa Fe Springs, CA, USA.
Methods Mol Biol. 2013;936:279-94. doi: 10.1007/978-1-62703-083-0_22.
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.
转基因动物模型是在体内测试基因功能和药物作用机制的宝贵工具。它们也是用于疾病病因和病理研究的最接近人体的模型。所有药物在获得美国食品药品监督管理局批准用于临床试验之前，必须在动物身上证明其安全性和有效性。为此，疾病转基因动物模型充当了药物评估的第一线。然而，目前尚无用于微小RNA（miRNA）研究的转基因动物模型。miRNA是一种小的单链调节RNA，能够沉默细胞内与miRNA具有完全或部分互补性的基因转录本（mRNA），可用于设计针对癌症多态性和病毒突变的新疗法。最近，在几乎所有真核生物中都发现了多种天然miRNA来源于发夹状RNA前体，包括酵母（粟酒裂殖酵母）、植物（拟南芥属）、线虫（秀丽隐杆线虫）、果蝇（黑腹果蝇）、鱼类、小鼠和人类，涉及细胞内对病毒感染的防御以及发育过程中某些基因表达的调控。为了促进体内miRNA研究，我们开发了一种先进的转基因策略，利用内含子miRNA在斑马鱼、鸡和小鼠中沉默特定基因。通过将发夹状前体miRNA结构插入基因的内含子区域，我们发现成熟miRNA由RNA聚合酶II（Pol II）成功转录，与编码基因转录本共表达，并通过天然RNA剪接和加工机制从编码基因转录本中切除。结合逆转录病毒转染系统，进一步测试设计的发夹状前体miRNA构建体插入细胞基因的内含子区域，以实现由基因启动子调控的组织特异性表达。由于逆转录病毒载体随机整合到宿主细胞基因组中，因此可以选择最有效的转基因动物并繁殖成为稳定的转基因品系以供未来研究。在这里，我们首次展示了使用先前描述的内含子miRNA表达系统生成转基因样动物模型，该系统已被证明对miRNA研究和miRNA相关靶基因功能的体内评估均有用。
