miRZip anti-sense microRNAs are stably expressed RNAi hairpins that have anti-microRNA activity. These miRZip shRNAs produce short, single-stranded anti-microRNAs that competitively bind their endogenous microRNA target and inhibit its function. The result is the derepression and elevation of the protein levels of the transcripts targeted by the microRNA being "zipped".
Permanent microRNA Knockdown
The miRZip short hairpin RNAs are cloned into SBI's pGreenPuro™ shRNA expression lentivector. The miRZip hairpins are rationally designed for asymmetry such that the upper strand of the hairpin (in gray) does not contain the endogenous microRNA sequence and the lower strand is preferred for producing anti-sense microRNAs (in green) that are fully complementary to a specific microRNA target.
miRZip lentivector constructs can be used for both GFP sorting and Puromycin selection for stable cell lines.
HEK-293 cells were transfected with a miRZip™ anti-microRNA expression vector, and puromycin (8-50 ug/ml final concentration) was then added to the cells 24 hours after transfection. The pictures were taken 24 hour after the initiation of the puromycin treatment. The control cells showed no survival after puromycin selection.
Thoroughly Understand MicroRNA Function SBI’s microRNA interference Lentivector System provides a convenient and effective approach to create stable cell lines or transgenic animals since it efficiently integrates the anti-microRNA expression construct of your choice into genomic DNA. Assays with cell lines or transgenic animals that permanently and heritably maintain a microRNA-specific suppression phenotype enable you to more thoroughly analyze the specific effects resulting from inhibiting your target microRNA.
Unique, Highly Effective System Our FIV and HIV-based Cloning and Expression Lentivectors are derived from the next generation of self-inactivating lentiviral vectors. This unique system offers a highly effective and very safe approach for introducing and expressing any anti-microRNA sequence in nearly any mammalian cell system.
Single Promoter Format The pGreenPuro™ siRNA/anti-microRNA lentivector employs a CMV promoter for the expression of dual copGFP and Puromycin markers and an H1 promoter for the expression of high levels of anti-microRNAs in the target cell.
Complete Lentivector Packaging System In order to stably transduce cells with SBI’s lentivectors, we recommend using SBI’s pPACK-H1 Lentivector Packaging System (cat.#LV500A-1) together with SBI's 293TN cell line (cat.#LV900A-1). The packaging system provides all the necessary coat proteins to manufacture pseudoviral particles for delivery of the anti-microRNA expression construct into any mammalian cells or model organisms, such as transgenic mice.
miRZip constructs express high levels of anti-microRNAs
MiRZip constructs were tested for anti-microRNA expression individually by transfecting HEK293 cells with 3ug miRZip plasmid DNA per well in 6 well tissue culture dishes using Lipofectamine 2000. The cells were lysed and total RNA extracted after 48 hours. The RNA samples were converted to cDNA using SBI's QuantiMir RT system for qPCR analysis. The levels of anti-microRNA expression were measured using QuantiMir primer assays designed for the specific miRZip anti-microRNA tested and compared to untransfected controls.
Expression validation of miRZip anti-microRNAs by qPCR
The QuantiMir cDNA from the transfections was diluted 1:50 and one microliter was used as template in 6ul qPCR reactions along with the primer assay for the specific anti-microRNA sequence labeled below. Standard thermal cycling conditions were used and data graphed as miRZip expression level compared to untransfected controls.
Modulation of miR-29a Target Protein levels using SBI's Lenti-miR-29a and miRZip-29a microRNA constructs
U937 Human leukemic monocyte lymphoma cell lines were transduced with control lentivirus (Control), Lenti-miR-29a (Overexpress) or miRZip-29a (Knockdown). The overexpression of miR-29a downregulated Target Protein levels and the suppression of endogenous miR-29a with miRZip-29a significantly de-repressed (upregulated) Target Protein levels.
Western Blot analysis of Target Protein levels
The higher the levels of microRNA-29a, the lower the levels of Target Protein and vice versa.
Suppression of cell invasion and tumor metastasis by miRZip-21 and increased metastasis using miRZip-145
To investigate the roles of miR-21 (oncogenic microRNA) and miR-145 (tumor suppressor microRNA) in breast cancer, cell invasion assays of MDA-MB-231 breast cancer cells were performed after lentiviral transduction with control vector lentivirus (Control) or miRZip-21 or miRZip-145 lentivirus. Matrigel chamber assays were then used to screen for invasive phenotypes.
After 20 h, invasion cells attached to the lower surface were fixed, followed by staining with 0.05% crystal violet. The number of invaded cells on the membrane was then counted under a microscope. Representative fields of invasive cells on the membrane are shown at the bottom for the Control and miRZip-21 or miRZip-145 treated cells.
The miRZip-21 lentivirus expressing anti-mir-21 small RNAs inhibited metastasis by 80% and the miRZip-145 lentivirus expressing anti-miR-145 RNAs increased metastasis by 60%. Additionally, the miRZip-145 lentivirus inhibited endogenous miR-145 and elevated protein expression levels of the miR-145 target oncogene c-Myc.
