pcDNA5/FRT/TOTOPO 载体简介 pcDNA5/FRT/TO-TOPO is a 5.2 kb expression vector designed to facilitate rapid cloning and tetracycline-regulated expression of PCR products using the Flp-In T-REx System (Catalog no. K6500-01) available from Invitrogen. When cotransfected with the pOG44 Flp recombinase expression plasmid into a Flp-In T-REx mammalian host cell line, the pcDNA5/FRT/TO-TOPO vector containing the PCR product of interest is integrated in a Flp recombinase-dependent manner into the genome. Expression of the gene of interest can then be induced by addition of tetracycline to the culture medium.
pcDNA5/FRT/TO, pcDNA5/FRT/TO-TOPO and pcDNA5/FRT/TO-E are designed for targeted Flp-In integration followed by tetracycline-regulated expression. Each vector contains a FRT site for efficient targeted integration and a hybrid CMV/TetO2 promoter for regulated expression. In addition, pcDNA5/FRT/TO-TOPO and pcDNA5/FRT/TO-E are adapted for TOPO-and Echo Cloning, respectively. TOPO Cloning enables rapid ligation of PCR products into pcDNA5/FRT/TO-TOPO in just five minutes on your bench top. Echo Cloning allows you to quickly shuttle your gene of interest into multiple expression vectors.
The Flp-In T-REx 293 Cell Line is available to save time in expression experiments. This cell line contains pFRT/lacZeo and pcDNA6/TR stably integrated and is tested for its ability to regulate expression . pcDNA5/FRT/TO-TOPO 载体含有以下元件:
A hybrid human cytomegalovirus (CMV)/TetO2 promoter for high-level, tetracycline-regulated expression of the gene of interest in a wide range of mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Hillen and Berens, 1994; Hillen et al., 1983; Nelson et al., 1987)
TOPO Cloning site for rapid and efficient cloning of Taq-amplified PCR products
FLP Recombination Target (FRT) site for Flp recombinase-mediated integration of the vector into the Flp-In T-REx host cell line
Hygromycin resistance gene for selection of stable cell lines (Gritz and Davies, 1983)
The control plasmid, pcDNA5/FRT/TO/CAT, is included for use as a positive control for transfection and expression in the Flp-In T-REx host cell line of choice. For more information about the Flp-In T-REx System, the pOG44 plasmid, and generation of the Flp-In T-REx host cell line, refer to the Flp-In T-REx Core Kit manual. 杂交 CMV/TetO2 启动子 Expression of your gene of interest from pcDNA5/FRT/TO-TOPO is controlled by the strong human CMV immediate early enhancer/promoter (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987) into which 2 copies of the tet operator 2 (TetO2) sequence have been inserted in tandem. Insertion of these TetO2 sequences into the CMV promoter confers regulation by tetracycline to the promoter.
The TetO2 sequences consist of 2 copies of the 19 nucleotide sequence, 5′-TCCCTATCAGTGATAGAGA-3′ separated by a 2 base pair spacer (Hillen and Berens, 1994; Hillen et al., 1983). Each 19 nucleotide TetO2 sequence serves as the binding site for 2 molecules of the Tet repressor. For more information about the mechanism of tetracycline regulation in the Flp-In T-REx System, refer to the Flp-In T-REx Core Kit manual. FRT 位点 The pcDNA5/FRT/TO-TOPO vector contains a single FRT site immediately upstream of the hygromycin resistance gene for Flp recombinase-mediated integration and selection of the pcDNA5/FRT/TO-TOPO construct following cotransfection of the vector (with pOG44) into a Flp-In T-REx 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 below. For more information about pOG44, refer to the pOG44 manual or the Flp-In T-REx Core Kit manual.
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 34 bp 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).
The hygromycin resistance gene in pcDNA5/FRT/TO-TOPO lacks a promoter and an ATG initiation codon; therefore, transfection of the pcDNA5/FRT/TO-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 T-REx 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/TO-TOPO at the FRT site. Flp 重组酶介导的DNA重组 In the Flp-InT-RExSystem, integration of your pcDNA5/FRT/TO-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 TOPO克隆工作原理 The plasmid vector, pcDNA5/FRT/TO-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 .
pcDNA5/FRT/TO-TOPO载体表达实验流程图
