BLOCK-iT Pol II miR RNAi Expression Vector Kits

Easily determine which cells are expressing your miRNA of interest by using the pcDNA6.2 GW/EmGFP-miR vector in the BLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP or the BLOCK-iT Lentiviral Pol II miR RNAi Expression System with EmGFP. Because the EmGFP is expressed co-cistronically with the miRNA of interest, it is possible to see up to 100% correlation of EmGFP expression with the knockdown activity of the miRNA (Figure 3).

Figure 3.EmGFP and miRNA expression. (A) Map of the Pol II miR RNAi expression cassette with EmGFP between Dra I restriction sites for easy removal. (B) Cells were transfected with mir RNAi targeting Lamin A/C using Lipofectamine 2000 reagent at an expected 50% efficiency. After 48 hours, cells were treated with Hoechst nuclear stain which stains all cells (left panel), stained with a red Lamin A/C stain, and monitored for GFP expression. Nearly half of the cells highly express the Lamin protein (center panel). When cells expressing EmGFP and Lamin A/C are overlayed, GFP expressing cells do not appear to have Lamin A/C present, and cells stained red for Lamin A/C do not appear to have any GFP expression. This suggests that cells expressing EmGFP are also all greatly reduced in Lamin A/C expression due to the presence of the miR RNAi construct that is co-cistronically expressed.

Our Pol II miR RNAi Expression Vectors provide an easy and effective way to get more than one knockdown in a single experiment. The Pol II promoter enables co-cistronic expression of multiple miRNAs allowing you to knockdown multiple targets from a single construct (Figure 4). Using a simple restriction enzyme procedure, you can link two or more miRNA sequences in any order. This is ideal for knockdown of more than one pathway component or splice variant in your cell type or for using knockdown to express synthetic phenotypes.

Most shRNA vectors are driven by Pol III promoters, which significantly limits the types of promoters that can be used in your RNAi experiments and makes tissue-specific expression in an in vivo system impossible. Other vector approaches use specially modified Pol II promoters, which cannot be easily exchanged. The BLOCK-iT Pol II miR RNAi Expression Vectors include the CMV immediate early Pol II promoter and are compatible with many other Pol II promoters (Figure 5) providing a flexible system to regulate knockdown or use promoters that are active in specific tissues of interest for in vivo studies.

Figure 5B and C. Knockdown of co-transfected lacZ and luciferase reporter genes in HepG2 cells using Multisite Gateway EmGFP-miR constructs with the α1 anti-trypsin promoter as the 5’ element and the polyA signal from the HSV thymidine kinase gene as the 3’ element.

Use the BLOCK-iT Inducible Pol II miR RNAi Expression Vector Kit with EmGFP to regulate RNAi experiments (Figure 6). Observe changes over time by controlling the initiation of the RNAi response with an inducible system. The kit contains the pT-REx-DEST30 Gateway vector which after simple cloning and shuttling techniques, produces a miR RNAi expression vector suitable for inducible knockdown. The pT-REx-DEST30 Gateway vector contains the CMV promoter with two copies of the tetracycline operator (tetO2) sequence allowing high-level and regulated expression. This permits the study of loss of function in a stably transfected cell line even if the gene of interest is essential. Also, induction of miR RNAi expression can be halted so phenotypic changes can be measured during recovery of gene function.

Gateway cloning offers a fast and efficient method to transfer a miRNA cassette into a wide selection of vectors using homologous recombination (Table 1). With the BLOCK-iT Pol II miR RNAi Expression Vector Kits, miRNAs are cloned directly into Gateway expression vectors, as opposed to Gateway entry vectors. As a result, there are two key differences between the pcDNA6.2-GW/miR and pcDNA6.2-GW/EmGFP-miR expression vectors and typical Gateway entry vectors (Figure 7):

1. miRNA expression vectors include the CMV promoter. Once your miRNA sequence is cloned, you can immediately transfect and get your miRNA expression.
2. attB sites flank the miRNA (and GFP sequences if using pcDNA6.2-GW/EmGFP-miR). For expression in different DEST (destination) vectors, the inserts must first be transferred to a pDONR (donor) vector and then to a DEST vector of choice by a dual Clonase reaction (pDONR 221 vector, BP and LR Clonase II Enzyme Mixes, and pLenti6/V5-DEST vector are provided in the BLOCK-iT Lentiviral Pol II miR RNAi Expression System).

You can avoid time-consuming and tedious subcloning procedures and easily move to as many DEST vectors as you want using Gateway recombination.

Our Lentiviral Pol II miR RNAi Expression Systems combine the advantages of the BLOCK-iT Pol II miR RNAi Expression Vector Kit with Virapower Lentiviral Expression Vectors, enabling stable delivery of engineered miRNAs into non-dividing, primary, and hard-to-transfect cells. For example, ViraPower Lentiviral Expression Vectors have been used to deliver miRNA sequences into HeLa cells to knockdown lamin A/C or lacZ (Figure 8). With this system, get long-term, stable expression of miRNA in the cell model system of your choice.

The HiPerform lentiviral Pol II vector (Figure 9) contains a mRNA stabilizing sequence (WPRE) and a nuclear import sequence (cPPT) which have generated up to 5-fold higher virus titers and EmGFP expression levels in many cell lines (Figure 10). Additionally, MultiSite technology allows you to express the EmGFP/miR RNAi cassette from CMV, EF-1a, or your own tissue-specific or other in vivo appropriate promoter.

• Achieve up to 5x higher titers (measured by GFP) allowing more cells to be transduced or higher multiplicities of infection (MOIs) to be employed
• Incorporate tissue-specific or another in vivo appropriate promoter
• Track miRNA expression through co-cistronic expression with EmGFP
• Knock down more than one gene simultaneously through expression of multiple miRNAs from a single transcript