The BLOCK-iT Pol II miR RNAi Expression Vector Kits combine the advantages of traditional RNAi vectors - stable expression and the ability to use viral delivery - with capabilities for tissue-specific expression and multiple target knockdown from the same transcript.
The vectors (Figure 1) included in the BLOCK-iT Pol II miR RNAi Expression Vector Kits and the BLOCK-iT Lentiviral Pol II miR RNAi Expression Systems are designed to express artificial miRNAs which are engineered to have 100% homology to your target sequence and will result in target cleavage. The BLOCK-iT Pol II miR RNAi Vector Kits offer:
- Easy hairpin design – BLOCK-iT miR RNAi hairpins targeting the majority of annotated human, mouse, and rat genes are ready to anneal and clone into either of the BLOCK-iT Pol II miR RNAi Expression Vectors. Just search for your gene!
- Easy expression tracking - Co-cistronic expression of Green Fluorescent Protein (GFP) and the miR-adapted shRNA of interest enables easy analysis and reliable correlation of reporter activity and knockdown.
- Multiple target knockdown - Co-cistronic expression enables simultaneous knockdown of multiple targets and the generation of synthetic phenotypes.
- Tissue-specific experimental options - Using a wide selection of destination vectors or Multisite Gateway applications you can add your promoter of choice for applications in vitro or in vivo.
- Inducible expression - Regulation of the RNAi response permits the study of changes over time, allows loss-of-function experiments to be performed, and provides an excellent control system where phenotypic changes can be measured during recovery of gene function.
- Gateway compatibility - A wide selection of our destination vectors can be used, including lentiviral vectors for stable expression in any cell type, such as primary and non-dividing cells.
BLOCK-iT Pol II miR RNAi Expression Vectors
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.
High success rate with BLOCK-iT Pol II miR RNAi Designer
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:
- Choose the miR RNAi design option.
- 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.
- 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.
- Cohesively ligate the duplex into the linearized vector.
Reliable tracking of your miR RNAi cassette
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.
Knockdown multiple targets with BLOCK-IT Pol II miR RNAi Expression Vectors
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.
BLOCK-iT Pol II miR RNAi Expression Vectors are compatible with multiple promoters
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.
Figure 5a. BLOCK-iT Pol II miR RNAi Expression Vectors are compatible with multiple promoters. Normalized reporter activities from lysates of GripTite 293 cells co-transfected with 100ng each luciferase and lacZ reporter plasmids and 300 ng of pcDNA 6.2-GW/EmGFP-miR (CMV) or pEF-GW/EmGFP-miR
(EF-1 a) expression vectors per well.
Inducible expression for control over your experiment
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 compatibility for a wide selection of experimental options
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):
- miRNA expression vectors include the CMV promoter. Once your miRNA sequence is cloned you can immediately transfect and get your miRNA expression.
- 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
|Destination vector or system||Compatibility||Cat. no.|
|ViraPower Lentiviral Vectors, including Multisite Gateway®||Compatible with pLenti6/V5-DEST|
|EF-1a promoter (pEF-DEST51)||Yes||12285-011|
|N-terminal reporter tags (GeneBLAzer™, YFP, Lumio™ )||Compatible with pcDNA 6.2/N-YFP/DEST||V358-20|
|Multisite Gateway (pDEST/R4-R3)||Compatible with TKpolyA 3’ element and various 5’ promoter elements||12537-023|
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.
BLOCK-iT Pol II miR RNAi Vector Kits include the pcDNA 6.2-GW/miR and pcDNA 6.2-GW/EmGFP-miR expression vectors. Cloning directly into a Gateway expression vector allows for immediate miRNA expression with the CMV promoter. To shuffle the miRNA cassette into other DEST vectors, use a BP Clonase II reaction followed by an LR Clonase II reaction or a BP/LR rapid reaction (see manual for details).
For Research Use Only. Not for use in diagnostic procedures.