简介 pcDNAFRTTO vector is a 5.1 kb inducible expression vector designed for use with the Flp-In T-REx System. The control plasmid, pcDNA5FRTTOCAT, is also included for use as a positive control for transfection and expression in your Flp-In T-REx host cell line of choice.
pcDNA5/FRT/TO is a 5.1 kb inducible expression vector designed for use with the Flp-In T-REx System (Cat. 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 vector containing the gene of interest is integrated in a Flp recombinase-dependent manner into the genome. Expression of the gene of interest may be induced by the addition of tetracycline to the culture medium. pcDNA5/FRT/TO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)
Multiple cloning site with 10 unique restriction sites to facilitate cloning the gene of interest
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 is controlled by the strong 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. 关于pcDNA5/FRT/TO需要注意的事项 The pcDNA5/FRT/TO 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 plasmid following cotransfection of the vector (with pOG44) into Flp-In T-REx mammalian host cells. 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.
The hygromycin resistance gene in pcDNA5/FRT/TO lacks a promoter and an ATG initiation codon; therefore, transfection of the pcDNA5/FRT/TO 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 at the FRT site. Flp 重组酶介导的DNA重组 In the Flp-In T-REx System, integration of your pcDNA5/FRT/TO inducible 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 (see below) 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 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
pcDNA5/FRT/TO 载体使用流程如下：
