microRNA Cloning Vectors

Express microRNA precursor transcripts using lentivectors

Efficient Delivery and Permanent microRNA overexpression
SBI’s microRNA Lentiviral Cloning and Expression System enables you to set up stable and heritable microRNA overexpression. You can easily and efficiently transduce dividing and quiescent mammalian cells—including difficult-to-transfect cells such as primary, stem, neuronal, and endothelial. In addition, our lentivectors can be used to generate microRNA overexpression in animals, such as transgenic mice.

Analyze the Specific Effects of microRNAs
The use of highly efficient and non-stressful lentiviral transduction greatly increases the likelihood that any observed phenotypic effects result from a specific microRNA.

Thoroughly Understand microRNA Function
The Lentivector system provides a convenient and effective approach to create stable cell lines or transgenic animals since it efficiently integrates the microRNA expression construct of your choice into genomic DNA. Assays with cell lines or transgenic animals that permanently and heritably maintain the overexpression phenotype.

Unique, Highly Effective System
SBI’s HIV and FIV-based Cloning and Expression Lentivectors are derived from self-inactivating lentiviral vectors. This unique system offers a highly effective and very safe approach for introducing and expressing any microRNA sequence in nearly any mammalian cell system.

Benefits and Features

End-User Applications

HIV-based lentivectors

 

Description	Vector Maps
High levels of microRNA precursor expression from CMV promoter. The multiple cloning site (MCS) located downstream of a CMV promoter allows for convenient cloning of your microRNA presursor of interest. The SV40 polyadenylation signal in both vectors enables efficient termination of transcription, resulting in high levels of steady-state expression. Downstream of your expression cassette is an EF1 promoter driving the expression of either the Puro, copGFP or RFP marker genes.
MicroRNA precursor genes are cloned into the 3' end of the copGFP or RFP transcript where they can be processed naturally into precursor and mature microRNAs. Positively expressing cells can be sorted using a GFP or RFP reporter.
FIV-based lentivectors

Description

Vector Maps

Express native microRNAs with the CMV promoter in target cells through transfection (plasmid form) or transduction (packaged constructs). —Multiple Cloning Site (MCS: for cloning the gene of interest in MCS located downstream of the WPRE element. —Polycistronic expression cassette includes copGFP fluorescent reporter and Zeocin resistance marker on the same transcript as the miRNA to enable tracking and selecting cells expressing the miRNA.

Online Resources

 

Technical References

Vector-based microRNA expression

Mathijs Voorhoeve, P., C.L. Sage, M. Schrier, A.J.M.Gillis, H. Stoop, R.Nagel, Y. Liu, J.V.Duijse, J. Drost, A. Griekspoor, E. Zlotorynski, N. Yabuta, G. D.Vita, H. Nojima, L.H.J.Looijenga, and R. Agami (2006). “A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors.” Cell, 124, 1169-1181

Shin, K. J., E. A. Wall, J. R. Zavzavadjian, L. A. Santat, J. Liu, J. I. Hwang, R. Rebres, T. Roach, W. Seaman, M. I. Simon and I. D. Fraser (2006). "A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression." PNAS U S A, 103(37): 13759-64.

Stegmeier, F., G. Hu, R. J. Rickles, G. J. Hannon and S. J. Elledge (2005). "A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells." PNAS U S A, 102(37): 13212-7.

Primary microRNA processing

Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. “The Drosha-DGCR8 complex in primary micorRNA processing”. Gene Develop, 18: 3016-3027, 2004.

Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT, Kim VN. “Molecular basis for the recognision of primary microRNAs by the Drosha-DGCR8 complex”. Cell, 125: 887-901.2006.

Zeng Y, Yi R, Cullen BR. “Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha”. EMBO J, 24:138-148, 2005.>

Zeng Y, Cullen BR. “Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences”. J Bio Chem, 280:27595-27603, 2005.

microRNA functional study

Bennasser, Y., S. Y. Le, M. L. Yeung and K. T. Jeang (2004). "HIV-1 encoded candidate micro-RNAs and their cellular targets." Retrovirology, 1(1): 43.

Esau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun Y, Koo S, Perera RJ, Jain R, Dean NM, Freier SM, Bennett CF, Lollo B, Griffey R. MicroRNA-143 regulates adipocyte differentiation. J Biol Chem. 2004 Dec 10;279(50):52361-5.

Hariharan, M., V. Scaria, B. Pillai and S. K. Brahmachari (2005). "Targets for human encoded microRNAs in HIV genes." Biochem Biophys Res Commun, 337(4): 1214-8.

Johnson, S. M., H. Grosshans, J. Shingara, M. Byrom, R. Jarvis, A. Cheng, E. Labourier, K. L. Reinert, D. Brown and F. J. Slack (2005). "RAS is regulated by the let-7 microRNA family." Cell, 120(5): 635-47.

Kim, V. N. (2005). "Small RNAs: classification, biogenesis, and function." Mol Cells, 19(1): 1-15.

Lee, R. C., R. L. Feinbaum and V. Ambros (1993). "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14." Cell, 75(5): 843-54.

Lee, Y., K. Jeon, J. T. Lee, S. Kim and V. N. Kim (2002). "MicroRNA maturation: stepwise processing and subcellular localization." Embo J, 21(17): 4663-70.

Olsen, P. H. and V. Ambros (1999). "The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation." Dev Biol, 216(2): 671-80.

Pfeffer, S., M. Zavolan, F. A. Grasser, M. Chien, J. J. Russo, J. Ju, B. John, A. J. Enright, D. Marks, C. Sander and T. Tuschl (2004). "Identification of virus-encoded microRNAs." Science, 304(5671): 734-6.

Yi, R., Y. Qin, I. G. Macara and B. R. Cullen (2003). "Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs." Genes Dev, 17(24): 3011-6.

microRNA expression vectors Quantity Product Size Catalog # Price pCDH-CMV-MCS-EF1-Puro cDNA Cloning and Expression Vector 10 ug CD510B-1 $423.50 pCDH-CMV-MCS-EF1-copGFP cDNA Cloning and Expression Vector 10 ug CD511B-1 $423.50 pCDH-CMV-MCS-EF1-RFP cDNA Cloning and Expression Vector 10 ug CD512A-1 $423.50 Ligase-Free miRNA Cloning and Expression (pPS-EF1-GFP-LCS) 20 reactions LF522A-1 $388.00 Ligase-Free miRNA Cloning and Expression (pPS-EF1-RFP-LCS) 20 reactions LF522B-1 $388.00 pMIF-cGFP-Zeo pre-miRNA Control Vector (FIV-based) 10 ug MIFCZ300PA-1 $367.50 Related Products Quantity Product Size Catalog # Price pPACKF1 FIV Lentivector Packaging Kit 200 ul LV100A-1 $423.50 LentiSuite For HIV-based System 10 Viral Productions LV300A-1 $1,094.50 LentiSuite For FIV-based System 10 Viral Productions LV300B-1 $1,094.50 pPACKH1 HIV Lentivector Packaging Kit 200 ul LV500A-1 $423.50 PEG-it Virus Precipitation Solution (100 mL aliquot) 100 mL LV810A-1 $253.50 PEG-it Virus Precipitation Solution (250 mL aliquot) 250 mL LV825A-1 $552.50 293TN Producer Cell Line 2 x 10^6 cells LV900A-1 $264.00 UltraRapid Lentiviral Titering Kit (Human Version) 42 individual titers LV960A-1 $446.25

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