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 HuSH-29 hairpin constructs, it is now possible to make discoveries at an accelerated pace and at a relatively low cost.
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, HuSH-29 has the advantages of improved efficacy and minimal interferon response.
HuSH-29 Algorithm Design
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
OriGene provides 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.
Individual Gene Knockdown
siRNA introduced into cells lacks the ability to replicate itself. The cells eventually lose these oligos due to dilution by cell division. In transient transfection, the effect of siRNA on the cell system occurs within a few days after transfection and lasts only a few days. In stable transfection, introduction of a plasmid expressing short hairpin RNA (shRNA) with a selection marker allows selection for cells that have continuous suppression of the gene through expression of the short hairpin construct. Once expanded, these cells permanently contain the correct construct and express the encoding shRNA. Investigators then perform functional assays to measure the effect of the gene knockdown. . Transient transfections of any RNAi suffice for short-term experiments, while long-term experiments require stable transfection of expression or viral vectors.
HuSH-29 is considered the RNA interference method of choice because it can be easily adapted to the conditions, either transient or stable, required for any individual experiment.
Some immortalized cells transfect readily with lipids or electroporation, while more fragile and hard-to-transfect cells require viral delivery.. Retroviral delivery of RNAi can provide sustained silencing. When cells are transfected with shRNA-encoding retroviral vectors and selected with antibiotic pressure, the shRNA expression cassette becomes stably integrated into the host genome. This is a major advantage over siRNA oligos, which only silence genes for five to seven days. Many assays require a longer time period to achieve the desired effect. Retroviruses integrate into host genome on their own and generate stable gene silencing cell lines with high efficiency.
HuSH Performance Guarantee
For all HuSH shRNA products, OriGene provides a performance guarantee. If a set of OriGene constructs fails to produce at least 70% gene expression knock down, our scientists will work with you to pinpoint the problem and, if necessary, OriGene will replace your HuSH constructs with another set against that target gene, free of charge.
Purified Plasmid DNA
For your convenience, each construct includes 5 ug of ready-to-use plasmid at no extra charge. With purified DNA you can time shift your experiments and verify insert sequence immediately. Supplying purified DNA saves time, stores conveniently, and reduces contamination risk.
HuSH products contain one type of control: negative scrambled control.