BLOCK-iT™ Pol II miR RNAi Expression Vector Kit with EmGFP

miRNAs are sometimes expressed in clusters in long primary transcripts driven by RNA Pol II (Lee et al., 2004). Our vectors support chaining of miRNAs to express them in one primary transcript, thus ensuring co-cistronic expression of multiple miRNAs. In the final construct, the original pattern of restriction sites is regenerated, making the construct amenable to multiples rounds of chaining. The figure below shows the principle of chaining two miRNAs, derived from two different miRNA vectors, into one miRNA expression vector. Note: Chaining together miRNAs targeting different genes usually results in slightly reduced knockdown of each gene. Chaining different miRNAs targeting the same gene or repeating one miRNA can enhance knockdown. Due to increased processing, EmGFP expression is attenuated by miRNA chaining. See page 33 of the manual for directions on how to chain pre-miRNAs.

The EmGFP from the pcDNA6.2-GW/EmGFP-miR expression vector has the following excitation and emission wavelengths, as published in the literature (Tsien, 1998): 487 nm and 509 nm, respectively. Detection can be performed using a standard FITC filter set. We recommend Omega XF100.

We would recommend use of our pcDNA6.2GW/EmGFP-miR vector, where EmGFP is expressed co-cistronically with your miRNA of interest. You should see 100% correlation of EmGFP expression with the knockdown activity of your miRNA.

The vectors are designed and optimized for expressing modified miR155 structure, and finally make siRNA for gene targeting using RNAi pathways. Additional optimization might be necessary to express native miRNA. Alternatively, the sequences of native miRNA can be cloned into a standard protein expression vector and inspect for miRNA production. Please see our two suggestions for miRNA overexpression:

1. Use PCR on gDNA to amplify the endogenous pre-miRNA hairpin as well as ~50-80 bp of flanking sequences on each side, then TOPO clone into an expression vector such as one of our pcDNA vectors. This will produce a transcript which contains the pre-miRNA in the context of its natural flanking sequences. This will probably be the best mimic of the endogenous miRNA, because the flanking sequences and precursor have the information needed to correctly process out the mature miRNA. The disadvantage of this technique is that it is somewhat laborious and wouldn't be as amenable to looking at many miRNAs (each one requiring identification of the genomic locus, primer design, and successful PCR).
2. Use the mature miRNA sequence (or the first 21 nucleotides of it) as the “antisense” sequence in Invitrogen's BLOCK-iT Pol II miR RNAi vector system. This technique has been successfully published (see Lee et al., PNAS 2006;103;15669-15674) and is quick and simple for design and cloning.
Using this method, many miRNA vectors could be built the same way. We also have a fairly good understanding that the major product of the miR RNAi vectors has the expected 5' end for the mature miRNA. However, we also know that the 3' end is variable and includes a number of slightly smaller and longer species that can include nucleotides from the loop (GUU…). The 3' end is probably least critical to miRNA function, but there may be some miR:target interactions for which it is important, and we just don't know how closely these mimic endogenous miRNAs.

These vectors can be used for stable expression and the ability to use viral delivery. These miR RNAi 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. 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 kits offer over 70% knockdown success, easy expression tracking (with co-cistronic expression of Green Fluorescent Protein), multiple target knockdown, and constitutive or inducible expression.