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Abstract: MicroRNAs (miRNAs) are small endogenous RNAs molecules, approximately 21–23 nucleotides in length, which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction of eukaryotic miRNA expression vector was described. The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein (EGFP) gene was used as reporter gene. The Bombyx mori N (BmN) Cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

[1]Ambros, V., 2003. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell, 113(6):673-676.

[2]Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2):281-297.

[3]Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell, 136(2):215-233.

[4]Bejarano, F., Smibert, P., Lai, E.C., 2009. miR-9a prevents apoptosis during wing development by repressing Drosophila LIM-only. Dev. Biol., 338(1):63-73.

[5]Biryukova, I., Asmar, J., Abdesselem, H., Heitzler, P., 2009. Drosophila mir-9a regulates wing development via fine-tuning expression of the LIM only factor, dLMO. Dev. Biol., 327(2):487-496.

[6]Bushati, N., Cohen, S.M., 2007. microRNA functions. Annu. Rev. Cell Dev. Biol., 23(1):175-205.

[7]Cai, X., Hagedorn, C.H., Cullen, B.R., 2004. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA, 10(12):1957-1966.

[8]Cai, Y., Yu, X., Zhou, Q., Yu, C., Hu, H., Liu, J., Lin, H., Yang, J., Zhang, B., Cui, P., et al., 2010. Novel microRNAs in silkworm (Bombyx mori). Funct. Integr. Genomics, 10(3):405-415.

[9]Cao, J., Tong, C., Wu, X., Lv, J., Yang, Z., Jin, Y., 2008. Identification of conserved microRNAs in Bombyx mori (silkworm) and regulation of fibroin L chain production by microRNAs in heterologous system. Insect Biochem. Mol. Biol., 38(12):1066-1071.

[10]Chang, K., Elledge, S.J., Hannon, G.J., 2006. Lessons from Nature: microRNA-based shRNA libraries. Nat. Methods, 3(9):707-714.

[11]Chen, C., Ridzon, D.A., Broomer, A.J., Zhou, Z., Lee, D.H., Nguyen, J.T., Barbisin, M., Xu, N.L., Mahuvakar, V.R., Andersen, M.R., et al., 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic. Acids Res., 33(20):e179.

[12]Chen, C.Z., Li, L., Lodish, H.F., Bartel, D.P., 2004. MicroRNAs modulate hematopoietic lineage differentiation. Science, 303(5654):83-86.

[13]Du, G., Yonekubo, J., Zeng, Y., Osisami, M., Frohman, M.A., 2006. Design of expression vectors for RNA interference based on miRNAs and RNA splicing. FEBS J., 273(23):5421-5427.

[14]Feng, J., Wang, K., Liu, X., Chen, S., Chen, J., 2009. The quantification of tomato microRNAs response to viral infection by stem-loop real-time RT-PCR. Gene, 437(1-2):14-21.

[15]Filipowicz, W., Bhattacharyya, S.N., Sonenberg, N., 2008. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet., 9:102-114.

[16]Friedman, R.C., Farh, K.K., Burge, C.B., Bartel, D.P., 2009. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res., 19(1):92-105.

[17]Fukuda, Y., Kawasaki, H., Taira, K., 2006. Construction of microRNA-containing vectors for expression in mammalian cells. Methods Mol. Biol., 338:167-173.

[18]Gou, D., Zhang, H., Baviskar, P.S., Liu, L., 2007. Primer extension-based method for the generation of a siRNA/ miRNA expression vector. Physiol. Genomics, 31(3):554-562.

[19]Han, J., Lee, Y., Yeom, K.H., Nam, J.W., Heo, I., Rhee, J.K., Sohn, S.Y., Cho, Y., Zhang, B.T., Kim, V.N., 2006. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell, 125(5):887-901.

[20]He, P.A., Nie, Z., Chen, J., Lv, Z., Sheng, Q., Zhou, S., Gao, X., Kong, L., Wu, X., Jin, Y., et al., 2008. Identification and characteristics of microRNAs from Bombyx mori. BMC Genomics, 9(1):248.

[21]Hong, X., Hammell, M., Ambros, V., Cohen, S.M., 2009. Immunopurification of Ago1 miRNPs selects for a distinct class of microRNA targets. PNAS, 106(35):15085-15090.

[22]Hu, T., Fu, Q., Chen, P., Ma, L., Sin, O., Guo, D., 2009. Construction of an artificial microRNA expression vector for simultaneous inhibition of multiple genes in mammalian cells. Int. J. Mol. Sci., 10(5):2158-2168.

[23]Hu, T., Chen, P., Fu, Q., Liu, Y., Ishaq, M., Li, J., Ma, L., Guo, D., 2010. Comparative studies of various artificial microRNA expression vectors for RNAi in mammalian cells. Mol. Biotechnol., 46(1):34-40.

[24]Huang, Y., Zou, Q., Tang, S.M., Wang, L.G., Shen, X.J., 2009. Computational identification and characteristics of novel microRNAs from the silkworm (Bombyx mori L.). Mol. Biol. Rep., 37(7):3171-3176.

[25]Jagadeeswaran, G., Zheng, Y., Sumathipala, N., Jiang, H., Arrese, E.L., Soulages, J.L., Zhang, W., Sunkar, R., 2010. Deep sequencing of small RNA libraries reveals dynamic regulation of conserved and novel microRNAs and microRNA-stars during silkworm development. BMC Genomics, 11(1):52.

[26]Li, Y., Wang, F., Lee, J.A., Gao, F.B., 2006. MicroRNA-9a ensures the precise specification of sensory organ precursors in Drosophila. Genes. Dev., 20(20):2793-2805.

[27]Liu, J., 2008. Control of protein synthesis and mRNA degradation by microRNAs. Curr. Opin. Cell Biol., 20(2):214-221.

[28]Liu, S., Zhang, L., Li, Q., Zhao, P., Duan, J., Cheng, D., Xiang, Z., Xia, Q., 2009. MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori). BMC Genomics, 10(1):455.

[29]Liu, S., Li, D., Li, Q., Zhao, P., Xiang, Z., Xia, Q., 2010. MicroRNAs of Bombyx mori identified by Solexa sequencing. BMC Genomics, 11(1):148.

[30]Molnar, A., Bassett, A., Thuenemann, E., Schwach, F., Karkare, S., Ossowski, S., Weigel, D., Baulcombe, D., 2009. Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J., 58(1):165-174.

[31]Niu, Q.W., Lin, S.S., Reyes, J.L., Chen, K.C., Wu, H.W., Yeh, S.D., Chua, N.H., 2006. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat. Biotechnol., 24(11):1420-1428.

[32]Parizotto, E.A., Dunoyer, P., Rahm, N., Himber, C., Voinnet, O., 2004. In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev., 18(18):2237-2242.

[33]Park, W., Zhai, J., Lee, J.Y., 2009. Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes. Plant Cell Rep., 28(3):469-480.

[34]Pillai, R.S., Bhattacharyya, S.N., Filipowicz, W., 2007. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol., 17(3):118-126.

[35]Qiu, L., Wang, H., Xia, X., Zhou, H., Xu, Z., 2008. A construct with fluorescent indicators for conditional expression of miRNA. BMC Biotechnol., 8(1):77.

[36]Ritchie, W., Legendre, M., Gautheret, D., 2007. RNA stem-loops: to be or not to be cleaved by RNAse III. RNA, 13(4):457-462.

[37]Rumi, M., Ishihara, S., Aziz, M., Kazumori, H., Ishimura, N., Yuki, T., Kadota, C., Kadowaki, Y., Kinoshita, Y., 2006. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector. Biochem. Biophys. Res. Commun., 339(2):540-547.

[38]Sakurai, H., Izumi, S., Tomino, S., 1990. In vitro transcription of the plasma protein genes of Bombyx mori. Biochim. Biophys. Acta, 1087(1):18-24.

[39]Scherr, M., Eder, M., 2007. Gene silencing by small regulatory RNAs in mammalian cells. Cell Cycle, 6(4):444-449.

[40]Schmollinger, S., Strenkert, D., Schroda, M., 2010. An inducible artificial microRNA system for Chlamydomonas reinhardtii confirms a key role for heat shock factor 1 in regulating thermotolerance. Curr. Genet., 56(4):383-389.

[41]Shan, Z., Lin, Q., Deng, C., Li, X., Huang, W., Tan, H., Fu, Y., Yang, M., Yu, X., 2009. An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs. Mol. Biol. Rep., 36(6):1483-1489.

[42]Shan, Z., Lin, Q., Deng, C., Zhou, Z., Tan, H., Fu, Y., Li, X., Zhu, J., Mai, L., Kuang, S., et al., 2010. Comparison of approaches for efficient gene silencing induced by microRNA-based short hairpin RNA and indicator gene expression. Mol. Biol. Rep., 37(4):1831-1839.

[43]Shibata, A., Iwaki, A., Fukumaki, Y., 2007. A novel expression system for artificial miRNAs containing no endogenous miRNA precursor sequences. J. RNAi Gene Silencing, 3(1):237-247.

[44]Silva, J.M., Li, M.Z., Chang, K., Ge, W., Golding, M.C., Rickles, R.J., Siolas, D., Hu, G., Paddison, P.J., Schlabach, M.R., et al., 2005. Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet., 37:1281-1288.

[45]Stefani, G., Slack, F.J., 2008. Small non-coding RNAs in animal development. Nat. Rev. Mol. Cell Biol., 9(3):219-230.

[46]Sun, D., Melegari, M., Sridhar, S., Rogler, C.E., Zhu, L., 2006. Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown. Biotechniques, 41(1):59-63.

[47]Sun, H., Li, Q.W., Lv, X.Y., Ai, J.Z., Yang, Q.T., Duan, J.J., Bian, G.H., Xiao, Y., Wang, Y.D., Zhang, Z., et al., 2010. MicroRNA-17 post-transcriptionally regulates polycystic kidney disease-2 gene and promotes cell proliferation. Mol. Biol. Rep., 37(6):2951-2958.

[48]Tang, G., Tang, X., Mendu, V., Jia, X., Chen, Q.J., He, L., 2008. The art of microRNA: various strategies leading to gene silencing via an ancient pathway. Biochim. Biophys. Acta, 1779(11):655-662.

[49]Tong, C.Z., Jin, Y.F., Zhang, Y.Z., 2006. Computational prediction of microRNA genes in silkworm genome. J. Zhejiang Univ.-Sci. B, 7(10):806-816.

[50]Trujillo, R.D., Yue, S.B., Tang, Y., O′Gorman, W.E., Chen, C.Z., 2010. The potential functions of primary microRNAs in target recognition and repression. EMBO J., 29(19):3272-3285.

[51]Vaucheret, H., Vazquez, F., Crete, P., Bartel, D.P., 2004. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev., 18(10):1187-1197.

[52]Xu, J., Zeng, J.Q., Wan, G., Hu, G.B., Yan, H., Ma, L.X., 2009. Construction of siRNA/miRNA expression vectors based on a one-step PCR process. BMC Biotechnol., 9(1):53.

[53]Yu, X., Zhou, Q., Li, S.C., Luo, Q., Cai, Y., Lin, W.C., Chen, H., Yang, Y., Hu, S., Yu, J., 2008. The silkworm (Bombyx mori) microRNAs and their expressions in multiple developmental stages. PLoS One, 3(8):e2997.

[54]Yu, X., Zhou, Q., Cai, Y., Luo, Q., Lin, H., Hu, S., Yu, J., 2009. A discovery of novel microRNAs in the silkworm (Bombyx mori) genome. Genomics, 94(6):438-444.

[55]Zeng, Y., Cullen, B.R., 2005. Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences. J. Biol. Chem., 280(30):27595-27603.

[56]Zeng, Y., Wagner, E.J., Cullen, B.R., 2002. Both natural and designed microRNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell, 9(6):1327-1333.

[57]Zhang, Y., Zhou, X., Ge, X., Jiang, J., Li, M., Jia, S., Yang, X., Kan, Y., Miao, X., Zhao, G., et al., 2009. Insect-specific microRNA involved in the development of the silkworm Bombyx mori. PLoS One, 4(3):e4677.

[58]Zhou, H., Xia, X.G., Xu, Z., 2005. An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res., 33(6):e62.