OmicsLink shrna Clones guaranteed knockdown even in difficult-to-transfect cells

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1 OmicsLink shrna Clones guaranteed knockdown even in difficult-to-transfect cells OmicsLink shrna clone collections consist of lentiviral, and other mammalian expression vector based small hairpin RNA (shrna) clones against genome-wide target genes from human and mouse. A set of four expression constructs for every target gene ensures high knockdown efficiency with minimal off-target effects. GeneCopoeia provides four shrna constructs for every target gene and guarantees that at least one of the four will have a knockdown effect of 70% on corresponding gene expression as determined by qrt- PCR. Figure 1. Lentiviral expression vectorbased shrna clones with H1 or U6 promoter. Features and benefits shrnas of varying lengths (19 to 29 bases) were used for different genes to make shrna expression constructs that have high knockdown efficiency with minimal off-target effect. shrnas are cloned into lentiviral and other mammalian vectors with various promoters and reporter genes. Vector plasmid transfection and virus transduction efficiency are monitored by egfp or mcherry reporter protein. Stable cell line selection with puromycin and egfp or mcherry in the vector backbones enables studies of the effect of long-term expression suppression as well as that of transient suppression. Lentiviral vectors allow efficient transduction of shrnas into non-dividing and difficult-to-transfect cell cultures as well as conventional dividing cell lines. The expression cassettes of all shrna clones are fully sequenced including the promoter, sense and antisense target sequences, hairpin, termination and other linker sequences.

2 Figure 2. Mechanism of shrna vectormediated gene silencing OmicsLink shrna expression vectors Vector Promoter Selection Marker Reporter Gene Viral type psilv-h1 H1 Puromycin egfp Lenti psilv-u6 U6 Puromycin egfp Lenti psilv-mh1 H1 Puromycin mcherry Lenti psilv-mu6 U6 Puromycin mcherry Lenti psi-h1 H1 Puromycin egfp N/A psi-u6 U6 Puromycin egfp N/A psi-mh1 H1 Puromycin mcherry N/A psi-mu6 U6 Puromycin mcherry N/A Product guarantee The expression cassettes of all shrna clones are fully sequenced including the promoter, sense and antisense target sequences, hairpin, termination and other linker sequences. For human kinome shrna clones in psi-sh1 vector, we provide 1 to 3 clones for each gene, all of which have been validated to have a KD effect of 70% of the original level using the surrogate colorimetric alkaline phosphatase assay. For all other genes, lentiviral and mammalian vector-based shrnas, we provide four shrna clone constructs for each gene. We guarantee that at least one of the four shrna constructs will have a knockdown effect of 70% on corresponding gene expression as determined by qrt-pcr, provided that all verification experiments are conducted in strict adherence to the protocols and procedures provided by GeneCopoeia. After adequate trouble shooting procedure is completed and it is found that none of the four shrna constructs can achieve guaranteed level of knockdown effect, GeneCopoeia will supply a new set of 1 to 4 shrna constructs free of charge.

3 Special collection validated human kinome shrna clones GeneCopoeia offers a set of shrna clones against 350 human kinase genes offering a guarantee of knockdown effect. Additional features include: shrnas designed for high KD effect and low off-target effect. shrna target sequences provided in the mammalian expression vector, psi-sh1, containing an egfp gene driven by CMV promoter that can be used to measure transfection efficiency. Super H1 promoter drives shrna expression. The KD effect of the shrna clones in this collection has been validated (see figure 3). For validation assays, expression clones were constructed in the psicheck-ap vector that transcribes a chimeric mrna. This chimeric mrna contains both the targeted gene and a reporter gene that encodes secreted alkaline phosphatase (AP). The transcription level of targeted genes can be assayed by the enzymatic activities of AP as the result of unperturbed transcription and translation of this enzyme in the absence of shrnas. Upon co-transduction of a shrna clone and the corresponding psicheck-ap expression clone, the destruction of the targeted gene mrna by the targeting shrna results in the degradation of the AP gene and, therefore, translation reduction of AP. The KD effect can be quantified by the colorimetric AP assay, which is illustrated in figure 3. Figure. 3. Validation of shrnas against human kinome members using vector-mediated (psi-sh1) shrnas and CMV driven expression clones (in psicheck-ap) that contain target kinase ORFs and express alkaline phosphatase (AP) for colorimetric assays. A. In the absence of shrna, AP is expressed and its activity is quantified by colorimetric assay. B. Co-transfection of shrna and AP-ORF expression clones, chimeric mrna for AP-ORF is destroyed and, subsequently AP translation and its activity is reduced.

4 Applications Single gene down-regulation The knocking down (KD) effect of the shrna clones for a single gene can be studied and compared with that of a scrambled nucleotide control clone. Pathway analysis Genes have been grouped into various signal transduction, metabolic, and disease pathways and associations, as well as gene families and groups. By arraying the shrna clones of known pathway(s) in 96 or 384 well plates, the role for a group of genes can be studied in a pathway. Currently, we offer customers with a large collection of shrna clones against the human kinome which can be grouped according to the functionality of each kinase. Validation studies The OmicsLink expression ready ORF clones together with the same ORF clones that contain silent mutations in shrna target sequence regions can be used for shrna validation studies and gene/protein functional rescue studies for genes/proteins targeted by corresponding shrnas. Measurement of knockdown (KD) effect by vector mediated shrna clones qrt-pcr Targeting of the corresponding mrna by a shrna expression cassette leads to cleavage and degradation of the mrna and possible subsequent reduction of protein expression. qrt-pcr is recommended as the first step in measuring KD effect. We offer validated qrt-pcr primers for corresponding target genes (and any other published transcripts). Western blot analysis Protein down-regulation is required in most cases for gene KD studies which can be assessed by western blot analysis. We offer more than 2,500 monoclonal and polyclonal antibodies against human proteins for use in western blot analysis. Functional assays KD effect of shrnas can be studied by end-point biological and biochemical assays such as cell proliferation, colony formation, cell cycle analysis, and migration based on the potential role played by each gene /protein. Functional assays by a reporter gene/protein Alternatively, when the endogenous transcript level of gene of interest is low and/or when qrt-pcr and western blot analysis is not feasible or possible, KD effect can be measured by co-transduction of shrna clone with an expression clone plasmid which is transcribed into a chimeric mrna transcript consisting of a reporter gene and the target gene ORF. The reporter gene encodes a reporter protein enzyme that can be used in functional assays. The destruction of the target gene mrna by targeting shrna results in the degradation of the reporter gene and, therefore, translation reduction of reporter protein enzyme. The KD effect can be quantified by the reporter enzymatic assays. See figure 3 as an example of this validation method.

5 RNAi Technology RNAi generally refers to RNA interference, which is employed by host cells to cleave double strand RNA (dsrna) from invading microbes. Long dsrnas can silence the expression of target genes in a variety of organisms and cell types using the RNA interference pathway. The long dsrnas can be processed into nucleotide small interfering RNAs (sirnas) by an RNase III-like enzyme, Dicer. Then, the sirnas assemble into endoribonuclease-containing complexes called RNA-induced silencing complexes (RISCs). The sirna strands subsequently guide the RISCs to base-pair with target mrna molecules where they then cleave and destroy the cognate RNA (Figure. 4). This is a natural mechanism whereby host organisms can defend themselves against invading microbes which use dsrnas in their life cycles. sirnas were originally identified as intermediates in the RNAi pathway after induction by exogenous dsrna as mentioned above. While the biogenesis of endogenous sirna utilized for down-regulation of mrna in all species is largely unknown, scientists have applied the sirna mechanism to knock down gene expression in many experimental systems. The widely used method for gene knock down employs in vitro chemically synthesized dsrna molecules in the size of nucleotides with 3 overhangs. Upon being transduced into cells, they can be similarly processed into forming RISC-shRNA complexes for subsequent gene down-regulation. Since target sequences are chosen selectively and specifically for each transcript, these chemically synthesized sirnas become a very effective tool in down-regulating gene expression specifically in the field of reverse genetics (Figure. 4). Figure 4. The mechanism of RNA interference (RNAi).