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 pcDNA™6.2-GW/miR and pcDNA™6.2-GW/EmGFP-miR 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:

  • Over 70% knockdown success - Higher design predictability means you’ll screen fewer sequences than with other vector-based RNAi methods.
  • Easy expression tracking - Co-cistronic expression of Green Fluorescent Protein (GFP) and the microRNA (miRNA) 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-cell or for in vivo knockdown.
  • 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 Invitrogen destination vectors can be used, including lentiviral vectors for stable expression in any cell type, such as primary and non-dividing cells.
  • BLOCK-iT™ miR RNAi Select – Pre-designed BLOCK-iT™ miR RNAi Select 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.
  • Validated DuoPaks - As an alternative to designing your own miRNA vector construct, choose from over 50 pre-constructed, bench-tested BLOCK-iT™ Pol II miR Validated miRNA Vector DuoPaks guaranteed to knockdown specific kinase targets†.

Figure 1. The BLOCK-iT™ Pol II miR RNAi expression vectors.

BLOCK-iT™ Pol II miR RNAi Expression Vectors

The powerful new BLOCK-iT™ Pol II miR RNAi Expression Vectors offer significant advantages over the current short-hairpin RNA (shRNA) vector technology currently used for RNAi vector applications (Table 1). These new 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.

Figure 2. Expression of an miR sequence using the BLOCK-iT™ Pol II miR RNAi Expression Vector Kits.

Table 1. Comparison of RNAi vectors: shRNA vs. miRNA


shRNA miRNA
Typical knockdown success rate* ~50% >70%
Expression tracking No Yes
Multiple target knockdown No Yes
Tissue specific expression No Yes
Gateway® compatibility with most DEST vectors No Yes

*Rate at which constructs reduce mRNA expression >70%

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:

  • Go to the BLOCK-iT™ RNAi Designer and choose the miR RNAi Design option.
  • Input the accession number or the sequence of your target of interest and the Designer will automatically generate high probability DNA duplexes that once processed will have 100% homology to your target of interest.
  • Hybridize the oligos to form a 60-bp duplex with 4-nt 5’ overhangs.
  • Cohesively ligate the duplex into the linearized vector.

With the BLOCK-iT™ Pol II miR RNAi Designer, you’ll get maximal knockdown for a diverse range of target sequences.

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 3). This gives you the confidence in tracking the presence of your miRNA in any cell type.

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. Cells were specifically transfected with Lipofectamine™ 2000 reagent at an expected 50% efficiency to demonstrate the 100% tracking of EmGFP and miRNA expression. After 48 hours, cells were stained with Hoechst nuclear stain which stains all cells, stained with a red lamin stain, and monitored for GFP expression. Nearly half of the cells highly express the lamin protein. When cells expressing EmGFP and lamin A/C stained 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 miRNA 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.


Figure 4a. Example restriction digestion/ligation scheme for combining miRNAs from different vectors. Sal I (S), BamH I (B), Bgl II (Bg), and Xho I sites (X) around the miRNA flanking regions (bars) are indicated. By cloning the BamH I – Xho I fragment containing miRNA 1 into the Bgl II-Xho I fragment of the vector containing miRNA 2, a dual-miRNA plasmid is created. The original restriction site pattern is recreated (restriction sites between the miRNAs are destroyed), and additional miRNAs can be added in the same manner. Alternatively, miRNAs can be added in front of miRNA 1 by combining Sal I-Bgl II and Sal I-BamH I fragments.

Figure 4b.
Results of experiment co-transfecting luciferase and lacZ reporter plasmids with single- or dual-miRNA vectors with the indicated inserts. Luciferase and ß-galactosidase activities are normalized to the single (neg) or dual (neg/neg) miRNA negative control inserts, which form a pre-miRNA but are not predicted to target any known vertebrate genes. Knockdown is slightly attenuated in the dual-miRNA vectors but remains very potent at =90%.

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.

BLOCK-iT™ Pol II miR RNAi Expression Vectors are compatible with multiple promoters


Figure 5a. 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.

Figure 5b. Knockdown of pSCREEN-iT™/lacZ-AAK1 and luc reporters in T-REx™ 293 cells with and without induction with 1 µg/ml tetracycline at 30 ng pT-REx-GW/EmGFP-miR (CMV/TO) plasmid per well.

Figure 5c. Knockdown of co-transfected lacZ and luciferase reporter genes in HepG2 cells using Multisite Gateway® EmGFP-miR constructs with the a 1 anti-trypsin promoter as the 5’ element and the polyA signal from the HSV thymidine kinase gene as the 3’ element.

Inducible Expression for Control Over Your Experiment

The new BLOCK-iT™ Inducible Pol II miR RNAi Expression Vector Kit with EmGFP gives customers the ability to regulate RNAi experiments (see Figure 5B).  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 2). 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 6):

  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 2. Gateway® destination vector compatibility

Destination vector or system Compatibility Cat. no.
ViraPower™ Lentiviral Vectors, including Multisite Gateway® Compatible with pLenti6/V5-DEST™
pLenti6/UbC/V5-DEST™
V496-10
V499-10
K4934-00
EF-1a promoter (pEF-DEST51) Yes 12285-011
T-REx™ (pT-REx™-DEST30) Yes 12301-016
Flp-In™ (pEF5/FRT/V5-DEST™) Yes V6020-20
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

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).

Figure 6. Gateway® cloning overview

Combine with lentivirus for stable, long-term expression

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 7). With this system, you’ll get long-term, stable expression of your miRNA in the cell model system of your choice.


Figure 7. Effective long term knockdown of endogenous target using lentiviral delivery
Lentiviral transduction of miRNA expression cassettes. Western blot analysis of HeLa cell populations stably transduced for 22 to 33 days with lentiviral particles expressing EmGFP and lamin A/C (lamin) or lacZ-directed miRNAs (MOI = 20). The blot was probed with anti-lamin A/C (top half) or ß-actin (bottom half) antibodies.


Figure 1A - The new BLOCK-iT™ HiPerform™ Lentiviral POL II miR RNAi Expression System with EmGFP. The pLenti6.4/CMVor Ef-1a/V5-M5--GW/EmGFP-miR vector is driven by the CMV jpromotor, has the blasticidin resistance marker, and is available with co-cistronic EmGFP expression as a reporter.


Figure 1B - Higher fluorescence evident from pLenti6.4 constructs using the BLOCK-iT™ HiPerform™ Lentiviral POL II miR RNAi Expression System.  Images taken four days following tradsduction of GripTite™ 293 cells at a MOI of 3 with the respective viruses.

The HiPerform™ vector 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. 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 Fig
  • Incorporate your own tissue specific or other in vivo appropriate promoter 
  • Track miRNA expression through co-cistronic expression with EmGFP 
  • Knockdown more than one gene simultaneously through expression of multiple miRNAs from a single transcript

BLOCK-iT™ miR RNAi Select

Pre-designed BLOCK-iT™ miR RNAi Select hairpins targeting the majority of the human, rat, and mouse genes are ready to anneal and clone into either of the BLOCK-iT™ Pol II miR RNAi Expression Vectors. Designed using a unique set of parameters, including an advanced mismatch alignment search, these artificial miRNAs are both highly effective at knocking down the target mRNA and also highly specific. Order now using the BLOCK-iT™ RNAi Express search engine or find out more about BLOCK-iT™ miR RNAi Select.

Bench-tested BLOCK-iT™ Pol II miR Validated miRNA Vector DuoPaks
In addition to designing your own miR RNAi vector, you can choose validated vectors available to human kinases. Bench-tested by Invitrogen scientists, each BLOCK-iT™ Pol II miR Validated miRNA Vector DuoPak contains two independent GFP vectors with pre-miRNA inserts designed to non-overlapping target sites. These validated DuoPaks are designed to all known splice variants, have been proven to knock down their target by at least 70%, and are guaranteed*. The Validated DuoPaks offer a unique convenience, since two sequences are necessary to confirm the knockdown phenotype and to be compatible with publication requirements. More kinases and ultimately other key human pathway components will continue to be added, so check regularly.
Use the best RNAi vectors with the greatest flexibility
With the BLOCK-iT™ Pol II miR RNAi Expression Vector Kits, BLOCK-iT™ Lentiviral Pol II miR RNAi Expression Systems, and the BLOCK-iT™ Pol II miR Validated miRNA Vector DuoPaks, you’ll get everything you need to effectively complete all of your demanding vector-based RNAi experiments. Get the promoter and experimental flexibility conferred by a Pol II-driven system while saving time with more effective design and knockdown.

Let Invitrogen Custom Services do the work for you

Invitrogen offers RNAi Services for each and every step of the RNAi experimental workflow. If you would like us to design and clone your miR RNAi vectors in 2-3 weeks, you can now order this service online. If you are looking for more than just cloning services, our RNAi experts will assist you in designing a service to meet your specific needs. Whether you just need assistance in designing RNAi reagents or you are looking for a more expansive service such as HTP cloning, screening and Lentivirus production, we have the capabilities to accelerate your research. Contact us today to discuss how we can help you achieve results faster with the quality service and products you expect. BLOCK-iT™ Pol II miR RNAi Services

* 100% performance guarantee: When you purchase a BLOCK -iT™ Pol II miR Validated miRNA Vector DuoPak you will get at least 70% knockdown of the target mRNA with two independent plasmids containing pre-miRNA inserts designed to non-overlapping target sites when transfected at an efficiency of at least 80%, and when target mRNA is quantified at 48 hours post-transfection. Transfection efficiencies can be estimated by detecting EmGFP fluorescence in whole cells or by knockdown measurements achieved with the positive control vectors against endogenous or stably expressed target genes. We also recommend the use of the negative control included with each Validated Vector DuoPak.

Ordering information on the latest BLOCK-iT™ Pol II miR Validated miRNA Vector DuoPaks.

The new 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.

The HiPerform™ vector 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. 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
  • Knockdown more than one gene simultaneously through expression of multiple miRNAs from a single transcript