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          pCDH-MSCV-MCS-EF1-Puro

          價格:2000元

          聯系方式:I47-825O-882O

          買家導航

          載體基本信息

          出品公司: SBI
          載體名稱: pCDH-MSCV-MCS-EF1-Puro
          質粒類型: 慢病毒表達載體;cDNA表達載體;雙啟動子載體
          克隆方法: 多克隆位點,限制性內切酶
          啟動子: MSCV
          載體大小: --
          5' 測序引物及序列: MSCV: GGGGTACAGTGCAGGGGAAAGAAT(注意: 該引物是SBI公司特別使用的MSCV引物,與測序公司擁有的通用型MSCV引物MSCV: CCCTTGAACCTCCTCGTTCGACC 完全不同,請務必不要弄混!)
          3' 測序引物及序列: pCDH1-R:CCTTCTCTAGGCACCCGTTCAAT
          載體標簽:
          載體抗性: 氨芐青霉素(Ampicillin)
          篩選標記: 嘌呤霉素
          克隆菌株: E.coli cells (RecA-)   推薦: Stbl2 ,OmniMAX 2 T1R
          宿主細胞(系): 造血干細胞、胚胎干細胞
          備注: pCDH-MSCV-MCS-EF1-Puro慢病毒表達載體是基于HIV的慢病毒載體;
          用于cDNA表達和克;高效轉染 細胞,建立穩定細胞系;
          MSCV啟動子驅動目的基因的高水平表達,EF1a啟動子驅動報告基因的中等水平的表達。
          產品目錄號: CD710B-1
          穩定性: 穩表達
          組成型/誘導型: 組成型
          病毒/非病毒: 慢病毒(HIV)

          載體質粒圖譜和多克隆位點信息

          pCDH-MSCV-MCS-EF1-Puro載體圖譜

          載體簡介

          背景簡介:
          
          This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors, and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on the SBI website. Before using the reagents and material supplied with this system, please read the entire manual.
          
          基于HIV-1的pCDH 慢病毒載體特征:
          
           Multiple Cloning Site (MCS)—for cloning the gene of interest in the MCS located downstream of the CMV promoter.
           WPRE element—enhances stability and translation of the CMV-driven transcripts.
           SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts.
           Hybrid RSV/5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.
           Genetic elements (cPPT, gag, env, LTRs)—necessary for packaging, transducing, and stably integrating the vira expression construct into genomic DNA.
           SV40 origin—for stable propagation of the pCDH plasmid in mammalian cells.
           pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells.
           Ampicillin resistance gene—for selection in E.coli cells.
          
          pCDH 慢病毒表達載體的優勢:
          
          Lentiviral expression vectors are the most effective vehicles for the delivery and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols. However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.
          
          pCDH 慢病毒載體的包裝載體及細胞系
          
          The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transiently co-transfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system,please refer to the Lentivector Expression System user manual.
          
          啟動子的選擇:
          
          SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.
          
          The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.
          The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).
          
          The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.
          
          MSCV promoter is the 5’-LTR promoter of murine stem cell virus. When a portion of the U3 region of the 3’ HIV LTR was replaced with the U3 region of MSCV LTR, the resulted hybrid HIV/MSCV LTR has dramatically increased the transgene expression level in human CD34+ hematopoietic cells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acceptor sites derived from the lentiviral vector. Further studies found that additional CpG mutations in the MSCV LTR reduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV LTR. After integration into genomic DNA, 3’MSCV/LTR will replace the 5’LTR and provide a high level of expression of the target gene and reporter gene downstream.
          
          SBI第三代慢病毒載體
          
          SBI offers a third generation of the most popular HIV-1 based lentivector expression system which consists of three maincomponents:
          (1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)
          (2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)
          (3) A pseudoviral particle producer cell line (e.g., 293TN cells)
          
          2A Peptide-enabled dual expression system
          Coexpression of a reporter gene together with a gene of interest is a useful approach for selecting transfected or transduced cells. This is commonly achieved by using two independent internal promoters, such as CMV and EF1 in pCDH-CMV-MCSEF1- copGFP, or by linking two transgenes with an internal ribosomal entry site (IRES) element in a single bicistronic transcript. Many dual promoter pairs have shown a high level of expression of both transgenes in standard cell lines— however, promoter interference often occurs in some cell lines. There are also two main problems that limit the use of IRES: the large size and the imbalanced expression between the first and second cistrons (H. Mizuguchi, 2000; X.Yu, 2003).
          The “self-cleaving” 2A peptides have been used successfully to generate multiple proteins from a single promoter in many applications (P. de Felipe, 2004; M.J. Osborn, 2005; P. de Felipe, 2006). The 2A-like sequences exist in several  viruses  and are used to mediate protein cleavage from a single open reading frame. Through a ribosomal skip mechanism, the 2A peptide prevents normal peptide bond formation between the 2A glycine and the 2B proline without affecting the translation of 2B (M.L. Donnelly, 2001):
          
          SBI’s cDNA expression vectors incorporate the 2A-like sequence (T2A) from the insect virus Thosea asigna to mediate the coexpression of a reporter gene with the target cDNA. Reporter genes have been cloned at either the first or second positions, and we achieved high expression levels at both locations.
          
          

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