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HuSH shRNA pRS Vectors 
What is in the pRS shRNA expression plasmid?
What is the advantage of OriGene’s product over any other company’s shRNA product?
Your HuSH product is stated to be “locus specific”. How do I know that it will knock down the expression of my variant or isoform?
Can I screen all of the constructs provided in HEK293 cells, and then pick the most effective one for primary retroviral infections?
Can I get a HuSH construct against any species other than human or mouse?
Will your HuSH products work with any retroviral packaging cell line?
Why should I buy a pRS mammalian shRNA expression plasmid rather than a synthetic siRNA?
What is the U6 promoter?
What is the gene specific shDNA inserted downstream of U6 promoter?
How does the transcription terminate?
How does OriGene verify the shDNA sequences for specific genes?
How should I use the products?
How do I choose the right shRNA expression vector for gene silencing?
Can I select for pRS plasmid transfected cells?
How can I create a stable cell line with a functional shRNA expression vector?
What use does the empty pRS vector serve?
What use does the pRS-shGFP (29) non-effective plasmid (TR30003) serve?
What is the OriGene guarantee on the shRNA expression plasmids?
What is in the pRS shRNA expression plasmid?
Answer: The pRS plasmid was designed to effectively express the insert DNA sequences in transfected mammalian cells. The OriGene gene specific silencing plasmids contain a U6 promoter, a 29 base pair (bp) gene specific sequence, a 7 bp intervening sequence and another 29 base pair gene specific sequence (a reverse complement to the first one) and a transcription termination sequence. The expression of the insert sequence in mammalian cells results in the formation of a short hairpin RNA with a 29 bp stem and a 7-nucleotide loop. Such a structure has been shown to effectively knock down the expression of target genes.
OriGene's pRS plasmid has been shown to knock down the expression of green fluorescent protein, luciferase and the HER2 oncogene. On average, more than 25% of the shRNA tested are capable of inhibiting the target genes by 70% or more. In addition, the pRS plasmid contains a puromycin selectable marker and a pair of retroviral integration site flanking the gene silencing cassette and the selection marker. Both the selection marker and the retroviral integration functions have been validated.
What is the advantage of OriGene’s product over any other company’s shRNA product?
Answer: Our algorithm for designing the target sequences engineered into each HuSH construct is supported by published research to be highly effective. Additionally, the use of 29mer target sequences has been shown to be superior to 21mer sequences in knocking down gene expression. Finally, OriGene’s guarantee on these plasmids (see below) reassures its customers that OriGene stands behind the effectiveness of each HuSH product.
Your HuSH product is stated to be “locus specific”. How do I know that it will knock down the expression of my variant or isoform?
Answer: Unless stated otherwise, shRNA constructs were designed to be effective against most transcriptional variants at a particular gene locus. If you would like a knockdown construct against a specific transcriptional variant(s), OriGene can generate a custom HuSH product that will selectively knockdown only the specified variants.
Can I screen the all the constructs provided in HEK293 cells and then pick the most effective one for primary retroviral infections?
Answer: Absolutely. We recommend that you screen all the constructs individually to identify the most effective constructs. HEK293 cells are a convenient and easily transfectable system for screening all human shRNA constructs (use NIH3T3 for mouse shRNA constructs). Other cell lines can also be used for this purpose, so long as the transfection efficiency is high. Afterwards, the effective construct(s) can be used for retroviral infection of or for transfection into your target cells.
Can I get a HuSH construct against any species other than human or mouse?
Answer: OriGene can do any custom HuSH design and construction, regardless of species specificity. The only information you need to provide is the accession number of sequence you wish to target. We can offer you assistance in identifying the particular species’ sequence that is orthologous to a human sequence.
Will your HuSH products work with any retroviral packaging cell line?
Answer: Our pRS vector has been designed for viral packaging in most commercially available packaging cell lines. However, please make sure that the packaging line has not been previously transfected with plasmids containing a puromycin resistance cassette. Furthermore, you need to ensure that the chosen cell line’s viral particles are able to infect your target cell line (some cell lines have restricted species specificity). We suggest packaging lines such as PT67 (Clontech) or Phoenix (Orbigen) for use with our constructs.
Why should I buy a pRS mammalian shRNA expression plasmid rather than a synthetic siRNA?
Answer: The pRS plasmids offer customers an alternative strategy for the inhibition of target genes. There are several advantages to this vector based approach:
1. The duration of gene silencing can be longer than with the synthetic siRNA (even permanent silencing with stable transfection). RNAi constructs in a vector permit continous shRNA transcription.
2. When the transfection efficiency is limiting, one can select the plasmid-transfected cells with puromycin to ensure efficient gene silencing in the cells examined.
3. One can obtain stable cell lines through either plasmid transfection or viral infection via a packaging cell line.
4. The plasmids can be amplified and become an unlimited source for many experiments.
What is the U6 promoter?
Answer: The pRS plasmid contains the human U6 promoter that has been used extensively to express shRNAs in mammalian cells through RNA Poll III. This promoter has been validated in the pRS plasmid in gene knock down experiments.
What is the gene specific shDNA inserted downstream of U6 promoter?
Answer: The shDNA inserts were generated using a validated algorithm, oligo synthesis and subsequent cloning. All sequences of the shRNA expression cassettes were verified through DNA sequence analysis and matched against the target gene sequences. All sequences and information on the target genes is provided for users.
How does the transcription terminate?
Answer: A stretch of 6 thymidine nucleotides (dT6) follows immediately after the shRNA sequences. This dT6 leads to the termination of the transcription immediately following the shRNA sequences.
How does OriGene verify the shDNA sequences for specific genes?
Answer: All shRNA sequences in the expression vectors were verified through DNA sequence analysis and matched against the target genes.
How should I use the products?
Answer: OriGene recommends that the customer first transform an aliquot of each of the plasmids into competent E. coli cells and perform DNA mini-prep. The plasmids can then be transfected into a target cell line. After transfection, cell lysates can be obtained and used for western blot analysis with an antibody against the target protein to verify the functionality of the shRNA vectors, or RNA can be harvested from transfected cells and used in quantitative RT-PCR to determine the loss of gene expression.
How do I choose the right shRNA expression vector for gene silencing?
Answer: The functionally active shRNA expression plasmids need to be tested experimentally in transiently transfected cells, individually and in combination with one another to identify the most potent knock down strategy.
Can I select for the pRS plasmid transfected cells?
Answer: Yes. One day after transfection, the cells can be selected with culture media containing 0.5 - 1.0 ug/mL puromycin (Sigma).
How can I create a stable cell line with a functional shRNA expression vector?
Answer: Stable cell lines can be generated by two different methods. First, target cells can be transfected with the shRNA plasmid. One day after transfection, the transfected cells can be selected with 0.5 - 1.0 ug/ml puromycin for 1-2 weeks with passages as needed. Alternatively, retroviral packaging cell lines can be used to generate retroviruses for stable cell line generation. For example, the plasmid can be transiently transfected into the Phoenix helper free amphotropic packaging cells line. Two days after the transfection, the cell culture supernatant is collected, filtered and used to infect the target cell lines. Stable cell lines can be established following drug selection.
What use does the empty pRS vector serve?
Answer: For shRNA experiments, it is important to demonstrate that the effect of a specific shRNA expression vector is gene specific, and it is not due to the non-specific effect known as interferon response. The empty vector will serve as the first tier negative control in a gene specific knock down experiment using our HuSH product. This vector can also be used for cloning your own shRNA construct(s).
What use does the pRS-shGFP (29) non-effective plasmid (TR30003) serve?
Answer: To specifically rule out the potential non-specific effect induced by expression of HuSH product, OriGene provides customers with a second tier negative control (TR30003), which was constructed by cloning a non-effective shGFP sequence cassette into our pRS vector. The plasmid should serve as a negative control for gene specific knock down experiments and exclude any potential interferon response.
What is the OriGene guarantee on the shRNA expression plasmids?
Answer: OriGene guarantees that the sequences in the shRNA expression cassettes are sequence verified to correspond to the target gene with 100% identity. The pRS expression plasmid vectors have been validated in gene silencing studies against green fluorescent protein, luciferase and human HER2 oncogene. If none of the four OriGene constructs produce at least 70% gene expression knock down, our scientists will work with you to pinpoint the problem and, if it is determined that our constructs are at fault, OriGene will replace your HuSH constructs with another set against that target gene, free of charge.
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