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shRNA Constructs

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HuSH-29 Overview

Gene silencing through the use of shRNA has become a primary tool for characterizing gene involvement in disease states and interactive pathways. In the past, tools for inactivating or disrupting the function of genes were cumbersome and unreliable. Creating and mapping genetic mutations using classic genetic approaches has been costly and slow.
With the use of the lentiviral HuSH-29 hairpin constructs, it is now possible to make discoveries with all advantages of lentiviral delivery, like sustained expression and silencing in non-dividing and difficult-to-transfect cells.

The length and design of HuSH-29 hairpin expression clones is an important improvement over the use of traditional 21mer shRNA and siRNA designs. Longer shRNA constructs appear to enter the RNAi pathway more efficiently and result in much higher potency and specificity than shorter expressed RNAi forms. In most mammalian cells, long double-stranded RNA provokes an interferon response as part of an antiviral defense. This interferon response induces a global shutdown of protein synthesis, thus precluding the use of long double-stranded RNA for specific gene silencing. This obstacle can be overcome by using shRNA less than 30 base pairs in length, which evades the radar of the mammalian interferon response and initiates strong and specific gene silencing. By its optimal length of 29 bases, HuSH-29 has the advantages of improved efficacy and minimal interferon response.

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shRNA Video
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Double Gene Knockdown Experiment

Double Gene Knockdown Experiment
In this double knockdown experiment cells were co-transfected with HuSH-29 against RFP (GFP Vector) and HuSH-29 against GFP (RFP Vector). The corresponding controls were scrambled shRNA in the same vectors. Left panel: Controls. Right panel: HuSH-29 knockdown.

Vector pGFP-C-shLenti

pGFP-C-shLenti vector is a third generation lentivector which requires the viral components carried in other vectors to produce viral particles. There are three major functional elements within the 5'LTR and 3'LTR: a shRNA expression cassette driven by an U6 promoter, a puromycin resistant gene driven by a SV40 promoter and a tGFP gene driven by a CMV promoter. All of them can be packaged to viral particles and transduced to many cell lines. The bacterial selection marker for the vector is Chloramphenicol.

Sequence  Datasheet

For our RNAi products, the shRNA expression cassette consists of a 29 bp target gene specific sequence, a 7 bp loop, and another 29 bp reverse complementary sequence, all under human U6 promoter. A termination sequence (TTTTTT) is located immediately downstream of the second 29 bp reverse complementary sequence to terminate the transcription by RNA Pol III. The gene-specific shRNA cassette is sequence-verified to ensure its match to the target gene.

Most RNAi companies have ready-made constructs that are often dated in the technology used to create them. OriGene uses a proven 29-mer short hairpin design, which has been demonstrated to be more effective than 21-mer versions. Steps have been taken to continually improve and optimize the algorithm used in sequence selection for HuSH-29 shRNA products.

  • Avoidance of sequences with three G’s, three C’s, four A’s and four T’s
  • GC content targeted at 50%, and must be between 30% and 70%
  • Selection of sequences in which the GC content is higher at 5’ than at 3’
  • Avoidance of internal palindrome sequences
  • Sequences are specific for locus ID and target to as many transcriptional variants of that locus as possible
  • Target sequences matching both human and mouse transcripts whenever it is possible
We provide 4 purified plasmid constructs, which can be used alone or in combination for transient or stable gene silencing studies. In addition to the rigorous attention that goes into the design, OriGene backs HuSH-29 products with a performance guarantee to ensure your success.

OriGene's non-proprietary pRS, pGFP-V-RS or pRFP-C-RS vectors allow both transient and stable transfection, as well as stable delivery of the shRNA expression cassette into host cells via a replication-deficient retrovirus. The pGFP-V-RS and pRFP-C-RS vector offers additional feature of turbo-GFP and turbo-RFP expression that facilitates easy monitoring of single or dual-gene transfection.

Features pRS pB-RS pGFP-V-RS pRFP-C-RS pGFP-B-RS
Fluorescent Label N/A N/A Green Red Green
E.coli Selection Ampicillin Ampicillin Kanamycin Chloramphenicol Kanamycin
Mammalian Selection Puromycin Blasticidin Puromycin Puromycin Blasticidin
Transient AND Stable
Yes Yes Yes Yes Yes
Viral Packaging Retroviral Retroviral Retroviral Retroviral Retroviral
Sequence (DOC)


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