Abstract
Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude of a human body for etiological and pathological research of diseases. All pharmaceutically developed drugs must be proven 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 human diseases serve as a front line for 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 that contain either complete or partial complementarity to the miRNAs, are useful for the design and development 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), 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 stateof-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 polymerase (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 hairpin-like pre-miRNA construct was further inserted into the intron 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 genes.
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References
Lin, S. L. and Ying S. Y. (2004) Novel RNAi therapy-intron-derived microRNA drugs. Drug Design Reviews 1, 247–255.
Tuschl, T. and Borkhardt A. (2002) Small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy. Mol. Interv. 2, 158–167.
Nelson, P., Kiriakidou M., Sharma A., Maniataki E., and Mourelatos Z. (2003) The microRNA world: small is mighty. Trends Biochem. Sci. 28, 534–539.
Ying, S. Y. and Lin S. L. (2005) Intronic microRNA (miRNA). Biochem. Biophys. Res. Commun. 326, 515–520.
Hall, I. M., Shankaranarayana G. D., Noma K., Ayoub N., Cohen A., and Grewal S. I. (2002) Establishment and maintenance of a heterochromatin domain. Science 297, 2232–2237.
Llave, C., Xie Z., Kasschau K. D., and Carrington, J. C. (2002) Cleavage of Scarecrowlike mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 2053–2056.
Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B., and Bartel, D. P. (2002) Prediction of plant microRNA targets. Cell 110, 513–520.
Lee, R. C., Feibaum, R. L., and Ambros, V. (1993) The C. elegans heterochromic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.
Reinhart, B. J., Slack, F. J., Basson, M., et al. (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906.
Lau, N. C., Lim, L. P., Weinstein, E. G., and Bartel, D. P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862.
Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B., and Cohen, S. M. (2003) Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25–36.
Xu, P., Vernooy, S. Y., Guo, M., and Hay, B. A. (2003) The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr. Biol. 13, 790–795.
Lagos-Quintana, M., Rauhut, R., Meyer, J., Borkhardt, A., and Tuschl, T. (2003) New microRNAs from mouse and human. RNA 9, 175–179.
Mourelatos, Z., Dostie, J., Paushkin, S., et al. (2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–728.
Zeng, Y., Wagner, E. J., and Cullen, B. R. (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333.
Hirose, Y. and Manley, J. L. (2000) RNA polymerase II and the integration of nuclear events. Genes Dev. 14, 1415–1429.
Kramer, A. (1996) The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367–409.
Miyagishi, M. and Taira, K. (2002) U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497–500.
Lee, N. S., Dohjima, T., Bauer, G., et al. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500–505.
Paul, C. P., Good, P. D., Winer, I., and Engelke, D. R. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505–508.
Xia, H., Mao, Q., Paulson, H. L., and Davidson, B. L. (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 1006–1010.
McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., and Kay, M. A. (2002) RNA interference in adult mice. Nature 418, 38–39.
Gunnery, S., Ma, Y., and Mathews, M. B. (1999) Termination sequence requirements vary among genes transcribed by RNA polymerase III. J. Mol. Biol. 286, 745–757.
Schramm, L. and Hernandez, N. (2002) Recruitment of RNA polymerase III to its target promoters. Genes Dev. 16, 2593–2620.
Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., and Williams, B. R. (2003) Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839.
Lin, S. L. and Ying, S. Y. (2004) Combinational therapy for HIV-1 eradication and vaccination. Intl. J. Oncol. 24, 81–88.
Stark, G. R., Kerr, I. M., Williams, B. R, Silverman, R. H., and Schreiber, R. D. (1998) How cells respond to interferons. Annu. Rev. Biochem. 67, 227–264.
Lin, S. L. and Ying, S. Y. (2004) New drug design for gene therapy-taking advantage of introns. Lett. Drug Design Discovery 1, 256–262.
Lin, S. L., Chang, D., Wu, D. Y., and Ying, S. Y. (2003) A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution. Biochem. Biophys. Res. Commun. 310, 754–760.
Lin, S. L., Chang, D., and Ying, S. Y. (2005) Asymmetry of intronic pre-miRNA structures in functional RISC assembly. Gene 356, 32–38.
Ambros, V., Lee, R. C., Lavanway, A., Williams, P. T., and Jewell, D. (2003) MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr. Biol. 13, 807–818.
Butz, S. and Larue, L. (1995) Expression of catenins during mouse embryonic development and in adult tissues. Cell Adhes. Commun. 3, 337–352.
Filipovska, J. and Konarska, M. M. (2000) Specific HDV RNA-templated transcription by pol II in vitro. RNA 6, 41–54.
Modahl, L. E., Macnaughton, T. B., Zhu, N., Johnson, D. L., and Lai, M. M. (2000) RNAdependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases. Mol. Cell Biol. 20, 6030–6039.
Abzhanov, A., Protas, M., Grant, B. R., Grant, P. R., and Tabin, C. J. (2004) Bmp4 and morphological variation of beaks in Darwin’s finches. Science 305, 1462–1465.
Wu, P., Jiang, T. X., Suksaweang, S., Widelitz, R. B., and Chuong, C. M. (2004) Molecular shaping of the beak. Science 305, 1465–1466.
Rodriguez, A., Griffiths-Jones, S., Ashurst, J. L., and Bradley, A. (2004) Identification of mammalian microRNA host genes and transcription units. Genome Res. 14, 1902–1910.
Clement, J. Q., Qian, L., Kaplinsky, N., and Wilkinson, M. F. (1999) The stability and fate of a spliced intron from vertebrate cells. RNA 5, 206–220.
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Lin, SL., Chang, SJ.E., Ying, SY. (2006). Transgene-Like Animal Models Using Intronic MicroRNAs. In: Ying, SY. (eds) MicroRNA Protocols. Methods in Molecular Biology™, vol 342. Humana Press. https://doi.org/10.1385/1-59745-123-1:321
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DOI: https://doi.org/10.1385/1-59745-123-1:321
Publisher Name: Humana Press
Print ISBN: 978-1-58829-581-1
Online ISBN: 978-1-59745-123-9
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