pcDNA5/FRT/V5-His-TOPO 载体 pcDNA5/FRT/V5-His-TOPOis a 5.1 kb expression vector designed to facilitate rapid cloning and expression of PCR products using the Flp-InSystem (Catalog nos. K6010- 01 and K6010-02) available from Invitrogen. When cotransfected with the pOG44 Flp recombinase expression plasmid into a Flp-Inmammalian host cell line, the pcDNA5/FRT/V5-His-TOPOvector containing the PCR product of interest is integrated in a Flp recombinase-dependent manner into the genome.
The pcDNA5/FRT/V5-His TOPOTA Expression Kit combines the Flp-InSystem with TOPOCloning technology to provide a highly efficient, rapid cloning strategy for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector for targeted expression of the gene of interest in mammalian cell lines. TOPO Cloning
requires no ligase, post-PCR procedures, or PCR primers containing special, additional sequences. pcDNA5/FRT/V5-His-TOPO载体含有以下元件: The human cytomegalovirus (CMV) immediate-early enhancer/promoter for highlevel constitutive expression of the gene of interest in a wide range of mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987)
TOPOCloning site for rapid and efficient cloning of Taq-amplified PCR products
C-terminal peptide containing the V5 epitope and a polyhistidine (6xHis) tag for detection and purification of recombinant protein
FLP Recombination Target (FRT) site for Flp recombinase-mediated integration of the vector into the Flp-Inhost cell line
Hygromycin resistance gene for selection of stable cell lines (Gritz and Davies, 1983)
The control plasmid, pcDNA5/FRT/V5-His/CAT, is included for use as a positive control for transfection and expression in the Flp-In host cell line of choice.
For more information about the Flp-In System, the pOG44 plasmid, and generation of the Flp-In host cell line, refer to the Flp-In System manual. The Flp-In System manual is supplied with the Flp-In Complete or Core Systems, but is also available for downloading from our World Wide Web site or by contacting Technical Service Flp 重组酶介导的DNA重组 In the Flp-In System, integration of your pcDNA5/FRT/V5-His-TOPO expression construct into the genome occurs via Flp recombinase-mediated intermolecular DNA recombination. The hallmarks of Flp-mediated recombination are listed below.
Recombination occurs between specific FRT sites on the interacting DNA molecules
Recombination is conservative and requires no DNA synthesis; the FRT sites are preserved following recombination and there is minimal opportunity for introduction of mutations at the recombination site
Strand exchange requires only the small 34 bp minimal FRT site FRT 位点 The FRT site, originally isolated from Saccharomyces cerevisiae, serves as a binding site for Flp recombinase and has been well-characterized (Gronostajski and Sadowski, 1985;Jayaram, 1985; Sauer, 1994; Senecoff et al., 1985). The minimal FRT site consists of a 34bp sequence containing two 13 bp imperfect inverted repeats separated by an 8 bp spacer that includes an Xba I restriction site. An additional 13 bp repeat is found in most FRT sites, but is not required for cleavage (Andrews et al., 1985). While Flp recombinase binds to all three of the 13 bp repeats, strand cleavage actually occurs at the boundaries of the 8 bp spacer region (Andrews et al., 1985; Senecoff et al., 1985).
upstream of the hygromycin resistance gene for Flp recombinase-mediated integration and selection of the pcDNA5/FRT/V5-His-TOPO construct following cotransfection of the vector (with pOG44) into a Flp-In mammalian host cell line. The FRT site serves as both the recognition and cleavage site for the Flp recombinase and allows recombination to occur immediately adjacent to the hygromycin resistance gene. The Flp recombinase is expressed from the pOG44 plasmid. For more information about the FRT site and recombination, see the next page. For more information about pOG44, refer to the pOG44 manual or the Flp-In System manual.
The hygromycin resistance gene in pcDNA5/FRT/V5-His-TOPO lacks a promoter and an ATG initiation codon; therefore, transfection of the pcDNA5/FRT/V5-His-TOPO plasmid alone into mammalian host cells will not confer hygromycin resistance to the cells. The SV40 promoter and ATG initiation codon required for expression of the hygromycin resistance gene are integrated into the genome (in the Flp-In host cell line) and are only brought into the correct proximity and frame with the hygromycin resistance gene through Flp recombinase-mediated integration of pcDNA5/FRT/V5-His-TOPO at the FRT site. For more information about the generation of the Flp-In host cell line and details of the Flp-In System, refer to the Flp-In System manual. TOPO克隆原理 The plasmid vector, pcDNA5/FRT/V5-His-TOPO, is supplied linearized with:
Single 3′ thymidine (T) overhangs for TA Cloning
Topoisomerase covalently bound to the vector (this is referred to as “activated” vector) Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3′ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I.The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO Cloning exploits this reaction to efficiently clone PCR products.
