BLOCK-iT Pol II miR RNAi Expression Vector Kits

BLOCK-iT Pol II miR RNAi expression vectors (Figure 1) offer significant advantages over conventional short-hairpin RNA (shRNA) vectors. These novel vectors include flanking and loop sequences from an endogenous miRNA which directs the excision of the engineered miRNA from a longer Pol II transcript (pri-miRNA). When present in the nucleus, these vectors efficiently use the endogenous cellular machinery to process knockdown sequences that are specifically designed to have 100% homology to your target of interest and will result in target cleavage (Figure 2). In addition, the loop sequence has a unique restriction site, so that it can be linearized for more efficient sequencing, sometimes a challenge with standard shRNA hairpins.

The BLOCK-iT HiPerform Lentiviral Pol II miR RNAi Expression System with EmGFP is the most powerful and flexible RNAi vector we have offered to date. This technology combines high titers and maximum expression. The ability to incorporate custom promoters makes the system suitable for in vivo applications.

Figure 1. The BLOCK-iT Pol II miR RNAi expression vectors The pcDNA 6.2-GW/miR and pcDNA 6.2-GW/EmGFP-miR vectors are designed for high-efficiency processing by Dicer from a microRNA-adapted backbone to express RNAi constructs (e.g. shRNAs) as miRNAs which are engineered to have 100% homology to your target sequence to result in target cleavage and silencing.
 

The BLOCK-iT Pol II miR RNAi Designer maximizes your chance of success by identifying an optimal target site within a gene for an miRNA to induce gene knockdown. The Designer provides the sequences of two DNA oligos that you will need to hybridize and clone into the BLOCK-iT Pol II miR RNAi Expression Vector. The process for producing highly effective miRNA inserts is very simple:

1. Choose the miR RNAi design option.
2. Input the accession number or the sequence of your target of interest and the Designer will generate high-probability DNA duplexes which, once processed, will have 100% homology to your target of interest.
3. Each BLOCK-iT miR RNAi Select hairpin is provided as two DNA oligos in separate tubes, which are ready for annealing and cloning. Hybridize the oligos to form a 60-bp duplex with 4-nt 5’ overhangs.
4. Cohesively ligate the duplex into the linearized vector.

Explore the BLOCK-iT RNAi Designer tool

You can easily determine which cells are expressing your miRNA of interest. Just use the pcDNA6.2 GW/EmGFP-miR vector in the BLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP (Emerald Green Fluorescent Protein) or the BLOCK-iT Lentiviral Pol II miR RNAi Expression System with EmGFP. Because the EmGFP is expressed co-cistronically with your miRNA of interest, you will see essentially 100% correlation of EmGFP expression with the knockdown activity of your miRNA (Figure 3a, b).

Figure 3a. 100% correlation of EmGFP and miRNA expression. Map of the BLOCK-iT Pol II miR RNAi Expression cassette with EmGFP between Dra I restriction sites for easy removal.
 

Figure 3b. 100% tracking of EmGFP and miR RNAi expression.

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 combined, it is clear that cells expressing GFP 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 demonstrates that cells expressing EmGFP are also all greatly reduced in Lamin expression due to the presence of the miR RNAi construct that is co-cistronically expressed.

BLOCK-iT 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.

Pol II promoter flexibility allows tissue-specific expression
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 virtually any other Pol II promoter (Figure 5) providing a flexible system to regulate knockdown or use promoters that are active in specific tissues of interest for in vivo studies. Find out more details about our vectors in Chapter 8 of the RNAi Handbook.

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The BLOCK-iT Inducible Pol II miR RNAi Expression Vector Kit with EmGFP gives customers the ability to regulate RNAi experiments (see Figure 6). Now you can 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 Technology provides a fast and efficient method to transfer your 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.

Table 1. Gateway destination vector compatibility

Table 1. Various Gateway destination vectors have been functionally tested for compatibility with the BLOCK-iT Pol II miR RNAi Expression Vector Kits for easy transfer of your miRNA cassette using homologous recombination.

The BLOCK-iT and BLOCK-iT HiPerform Lentiviral Pol II miR RNAi Expression Systems combine all of 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, you’ll get long-term, stable expression of your miRNA in the cell model system of your choice.

The HiPerform 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 your own tissue specific or other 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