RESULTS: Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. Objective: To construct the specific stable expression and high efficiency small interfering RNA(siRNA) expression vector that can block DNMT1 gene function. The siRNA eukaryotic expression vector against BCR/ABL mRNA has been successfully conctructed,and effectively inhibits the expression of BCR/ABL in K562 cells, inhibite cell growth and induce cell apoptosis. Construction of retroviral siRNA delivery vector Schematic of genome structure. Methods: Using vector-based RNA interference technique, the authors constructed a vector to transcribe functional short interfering RNA (RNAi). The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. GenScript vector-based siRNA technology is an adaptation of GenScript gene synthesis technology to the rise of RNA interference (RNAi) as a powerful tool for gene function analysis and drug target validation. To address the question of how shRNA sequence correlates with knockdown efficacy, 27 shRNA vectors from 11 different genes were designed and constructed (Table 1).Target sequences were selected in the coding region of each gene and were designed to broadly conform to the seminal studies of sequence features for siRNA oligomer efficacy … June 2009 ; BMC Biotechnology 9(1):53; DOI: 10.1186/1472-6750-9-53. • shRNA requires an expression vector, whereas siRNA does not. Aim: To construct the vector for efficient expression of siRNA using pre-mir30 backbone. RNA interference (RNAi) is a sequence-specific gene silencing mechanism. Construction of siRNA expression vectors. MCS, multiple cloning site. 1 College of Agriculture, Ningxia University, Yinchuan, 750021, China. Cells, Vol. 2. OBJECTIVE: To construct eukaryotic expression vector of siRNA specific for BCR/ABL and to investigate the effect of recombinant plasmid on BCR/ABL and P210 protein expression in K562 cells. Ambion's Solution: Silencer™ siRNA Construction Kit ... to effectively elicit gene silencing can be readily cloned into a plasmid or viral vector to create an siRNA expression vector. One disadvantage of SECs is that they are not as easily transfected into cells as siRNAs. After the siRNA expression vector was transfected into the MCF-7 cells, the expression of XIAP gene was inhibited significantly (by 90%). • Unlike the gene suppression of siRNA, the suppression of shRNA is long lasting, and if it is inserted via an appropriate viral vector, it may produce permanent gene silencing effects. A. Mol. However, when a large genome … Methods Two pairs of oligonucleotide sequences were designed and synthesized according to … [Construction of Eukaryotic Expression Vector of siRNA Specific for BCR/ABL Fusion Gene and Its Effects on K562 Cells] Zhongguo Shi Yan Xue Ye Xue Za Zhi. two specific template vectors (Temp-1 and Temp-2; see Appendix-2). PGK, murine phosphoglycerate kinase promoter. 127-130 Construction of a Vector Generating both siRNA and a Fluorescent Reporter: a siRNA Study in Cultured Neurons Seung Yong Yoon, Jung Eun Choi, Onyou Hwang1, Hea Nam Hong, Heuiran Lee2, Yoo Kyum Kim2, Sung-Woo Cho1, Hyun Kim3, and DongHou Kim* Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea; Synthesizing the neck ring structure of DNA which is corresponding to siRNA sequence, and then it is integrated into lentivirus plasmid vector. [Construction of DNMT1 siRNA stable expressing vector and evaluation of its silenced efficiency in blocking gene expression]. Jie … By Aurore Stroobants, Lionel Tafforeau, Allan Guiguen, Damien Hermand and Jean Vandenhaute. The GenScript siRNA technology package includes siRNA design, siRNA vectors, and custom siRNA construction. [Construction of NF-kappaB siRNA expression vector]. These modifications must be carefully applied as reductions in off-target effects are sometimes accompanied by deceases in the overall potency of suppression. Although the vector-driven shRNA approach to RNAi does not permit specific chemical modification of the silencing construct, siRNA oligomers can be chemically modified in order to reduce direct off-target effects. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems. siRNA Vector Construction Order Form We will design, construct, and package siRNA-expressing vector constructs targeting your gene of interest. 2.Interference of lentivirus in the construction of plasmid vector. Hairpin induced gene silencing of CP gene RB 35S (+) CP frag. Abstract. Puro Lastly, different primer designing guidelines need to be followed for constructing 3, 4, or 5 siRNA sites (see Appendix-1). The standard molecular cloning methods were used to construct the vectors containing multiple siRNA expression cassettes with the oligonucleotides shown in Fig. An organism's genomic DNA is extracted and then digested with a restriction enzyme. Protocol for shRNA construction-I: PCR method Preparation of cloning vector: 1. After transfection by lipofectmine (TM) reagent, the treated cells were selected by G418. Packaging involves the preparation of competent virus from DNA vectors. Methods: By chemical synthesis method, pre-mir30 backbone introduced an appropriate restriction enzyme site for foreign shRNA inserting was cloned into an expressing vector containing U6 promoter. Results. Construction and Functional Verification of siRNA Eukaryotic Expression Vector Directed at the Follicular Inhibin Alpha Gene in Ye Mule Sheep . Three types of small interfering RNA (siRNA) targeting the mouse TNF-α gene were designed, synthesized and transfected into RAW264.7 cells. The siRNA expression vector can then be used for stable expression and long term studies. [Article in Chinese] Kurexi Y(1), Huang GH, Long M, He J, Nasier M, Xia YP, Hamulati W. Author information: (1)Department of Biochemistry and Molecular Biology, Xinjiang Medical University, Urumqi 830054, China. The cellular growth activities in the MCF-7 cells transfected with siRNA against XIAP plasmid decreased obviously. Construction of a set of vectors allowing inducible production of siRNA in Schizosaccharomyces pombe. Incubate 3 µg of shRNA cloning vector with 5 units (NEB) of EcoRI and 10 units of AgeI, (double digestion) in a reaction volume of 100 µl at 370C for overnight (using NEB #4 buffer). We synthesized shorter primers (< 50 nucleotides) to generate a linear expression … 3.Packaging and concentrated purification of lentiviral vectors. The vector carries the inducible tetracycline promoter/operator for the regulated expression of proteins, the ompA signal for periplasmic secretion of the recombinant protein, the Strep-tag® for C-terminal fusion to the recombinant protein and the Ampicillin Resistance cassette. Our proprietary shRNA testing reporter vector allows for cloning of both shRNA template downstream of the H1 promoter and target sequences at the 3′ end of the GFP reporter. The separated fragments can then be excised and cloned into the vector separately. Vector-mediated RNAi (DNA or virus) Strategies of expressing double-stranded RNA Pol II and Pol III (type III) e.g U6 prom Promoter spacer/ intron terminator AAA alternative: tandem promoters, opposing promoters. Ψ+, extended packaging signal. Construction of a genomic library involves creating many recombinant DNA molecules. For organisms with very small genomes (~10 kb), the digested fragments can be separated by gel electrophoresis. These vectors feature the same promoters, ampicillin resistance gene, and E. coli origins of replication as the pSilencer 2.0-U6 and pSilencer 3.0-H1 siRNA Expression Vectors, but have the added benefit of an antibiotic resistence gene. The transfer construct carries the gene to be transduced or “transgene”. Based on these findings, we developed a lentiviral reporter vector for identification of functional shRNA constructs (Figure 1). The transfer construct also carries the sequences which are necessary for the general functioning of the viral genome: packaging sequence, repeats for replication and, when needed, priming of reverse transcription. All plasmids were derived from pMSCVpuro (Clontech). Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. The silencing efficiency of a new siRNA expressing vector was detected by transfection and Western blot. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression … Optionally, we offer packaging services to produce lentiviral particles that may be used to directly transduce your cells of interest. Source; PubMed; Authors: Jun Xu. The present invention relates to construction of hepatitis B virus siRNA expression vector and its antiviral therapeutic application. To construct the small interfering RNA (siRNA) expression vector of carcino-embryonic antigen (CEA) and inhibit the expression of CEA in EC9706 cells by RNA interference. 1a and additional files (see Additional file 1). Construction of siRNA/miRNA expression vectors based on a one-step PCR process. The primer sequences of siRNA on the target gene are detailed in Table I. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. The inserted U6 promoter is illustrated in blue, while the control and anti-NDR hairpins are shown in red and green, respectively. Design and preparation of shRNA plasmids. Replication-defective vectors always contain a “transfer construct”. Construction of Inducible shRNA Expression Lentiviral Vector 1. Six pSilencer siRNA Expression Vectors with antibiotic selectable markers are now available from Ambion (click here for comparison chart). Preparation of the Destination Vector with BbsI Digestion 1) Set up BbsI digestion of the destination vector pSiEB in 100ul reaction system: We offer a range of vectors with different fluorescent and selection markers with which to make constructs. Designing shRNA sequence and insert into BglII and XbaI sites of pENTR4-H1tetOx1 Target sequence length would be 19 bp starting with A or G. It would be better to introduce mismatch mutation disperse at 3 sites with C to T or A to G in the sense strand (Avoid making TTTT or TTATT sequence). 1, pp. Most vector-based shRNA systems contain a selectable marker to allow for the elimination of cells that have not been successfully transfected or transduced, and maintenance of cells with sustained gene knockdown. Results: The successful construction of siRNA against XIAP plasmid was identified with sequencing. • shRNA can be used for long term knockdown while siRNA can be used only for short term knockdown of genes. The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the mouse tumor necrosis factor-α (TNF-α) gene. 18, No. We will synthesize the DNA to encode the desired hairpin, clone it, and sequence it to verify correct construction. Zengwen Huang 1,2, WuReliHazi Hazihan 2 *, Baheti Bodai 2, Kadyken Rizabek 3, Nuralieva Ulzhan 3, Omarova Karlygash 4, Juan Zhang 1 and Yaling Gu 1 * Author Affiliations. Current options for constructing shRNA vectors include the use of … spacer nptII LB (-)CP frag. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems. Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. You only provide the GenBank (or other public database) accession number to a gene sequence and choose one of our siRNA cloning and expression vectors. 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