This is a free resource for the scientific community that is compiled by addgene this page is informational only this vector is not available from addgene please contact the manufacturer for further details. An orange fluorescent protein with a large stokes shift. The green fluorescent protein gfp from aequorea victoria is a versatile reporter protein for. The integrity of the final pegfp c1 cyp26a1 construct was confirmed by sequence analysis. Molecular cloning and characterization of stamp2, an. The ecori and xhoidigested segment coding for the truncated mutant rip3 n223 was inserted into ecorisalidigested pegfp c1 to yield.
Tbusa, formerly known as clontech laboratories, inc. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The eukaryotic expression vector pegfp c1 bmp2 was originally generated and transfected into cos7 cells to explore the function of bmp in bone and cartilage development. For license information, please contact a licensing representative by phone at 650. Intensiometric biosensors visualize the activity of multiple small. The effects of the overexpressed bmp2 on the migration of cos7 cells and the underlying molecular mechanism were investigated. Identification and characterization of a ligandregulated. Deep sequencing reveals complex spurious transcription from. Rafused rac1 or cdc42 were pcramplified and fused to egfpc1 clontech vector after excision of egfp at the site of nhei and bamhi. Mammalian expression vector, adds cterminal gfp tag.
Gfprip111218was generated by digesting pegfp c1 rip11 with bamhi and religating the backbone. The influence of bmp2 on cell migration and cofilin activity was detected by cell scratch assay and western blotting. Construction of a eukaryotic expression vector for pegfpfst and. Expression of egfp and gfps65t in transfected chok1 cells. Restriction map and multiple cloning site mcs of pacgfp1c1. Hindiiibamhi fragment from pegfp c1 rab11fip2 into the hindiiibamhi site of pegfp c2 clontech. Construction of a eukaryotic expression vector pegfpc1. The duck pegfpfst vector was successfully constructed and was confirmed to. The xbai site is methylated in the dna provided by clontech. The vectors pgfpmutlc1 and pegfpc1 clontech each encode the gfpmutl variant. Bamhi fragment was ligated into the response plasmid, pegfp enhanced green fluorescent protein c1 clontech, palo alto, ca designated as pegfp c1 cyp26a1 plasmid. Benchling works best when using a supported browser. Am using clontech s pegfp c1 vector along with lipofectamine2000 on 293t cells.
Restriction map and multiple cloning site mcs of pegfpc1. Construction of a eukaryotic expression vector pegfpc1 bmp2 and its effect on cell migration by. This nes ar mutant was cloned into pegfp c1 to produce pegfp nes argplgs 762. For other reading frames, use pegfp n1 or pegfp n2. Stable transfection of pegfp n1mog plasmid to utilize in multiple sclerosis gene therapy. By using gene cloning technique, eukaryotic expression vector pegfp c1 was used to construct the murine b71 recombinant plasmid pegfp c1 b7. Recombinant plasmid was transfected into lm8 cells with liposome and was confirmed by. Feeding of m or pegfp c1 dna to pregnant mice doublestranded circular or ecorilinearized mmp18 dna termed m dna in this report or plasmid pegfp c1 dna clontech was fed to 3 to 6monthold c57bl6 pregnant mice. I ags cells were transiently transfected with pegfpc1 plasmid egfp, pegfpc1 containing y5. The results revealed that in the pegfp n1hper2 and the pegfp n1 group, large numbers of mg63 expressed gfp. The c2 domains of the class i rab11 family of interacting. I need to clone the cdna of a type ii transmembrane protein from tnf super family and i need to make sure that the produced protein will be expressed properly on the cell surface hek293 cells. Im trying to set up the transfection technique in my new lab.
Grb2 is a key mediator of helicobacter pylori caga protein activities. The eukaryotic expression vector pegfpc1bmp2 was originally generated and. Construction of eukaryotic expression vector containing b7. As a control vector, pcmsegfpcyp26a1 plasmid, which has separate transcription cassettes. Xba i and bcl i sites are methylated in the dna provided by clontech laboratories, inc.
The fulllength stamp2 orf from pcriitopostamp2 was fused in frame to the cterminus of gfp using the vector pegfp c1 clontech. Forprofit entities wishing to use this product are required to obtain a license from clontech. To construct plssmorange c1 plasmid, lssmorange was pcr ampli. Tokue is a global leader in biotechnology innovation, offering great benefits and applications to the biopharmaceutical and diagnostic industries as well as for biotechnology research communities. The recombinant pegfp c1 bmp2 was effectively expressed in cos7 cells. Construction of a eukaryotic expression vector for pegfp. The amplified fragment was digested with bglii and xhoi and then cloned into bgliixhoi sites of pegfp c1 clontech to generate pegfp c1 rip3. Gli3 mutations in human disorders mimic drosophila cubitus. Gli3 cdna from the initiator codon to codon 423 was cloned by excising a saci and psti fragment from egli3phs and ligating it into the saci and psti sites of pegfp c2 to generate plasmid egli3gcps.
How to obtain a stable transfectant aug022005 hi, all. The vector pegfp c1 has been already utilised in gene expression in cell culture model xu et al. At 48 h posttransfection, the cells were fixed with 4% paraformaldehyde in pbs, ph 7. Clontech takara download free trial get snapgene viewer. Without the addition of a functional promoter, this vector will not express. I know my ligated product is present via pcr and by simply running it on a gel. Egfp was expressed in 70% of cells in the pegfp n1hper2 group and 75% of cells in the pegfp n1 group, suggesting that pegfp n1hper2 and pegfp n1 may be effectively transfected into mg63 cells, resulting in a high level of egfp expression. The substituted nucleotides encoded the amino acid sequence gplgs. The resulting pcr product was cloned into the same sites in the vector pegfp c1 clontech. Description pegfp c1 encodes redshiftedvariant wildtypegfp whichhas been optimized brighterfluorescence higherexpression mammaliancells. Hi all, i am new to the forums, but i have been a longtime lurker when the need arose. This project is supported bytokuewhich specializes in manufacturing ultrapure antibiotics for a broad spectrum of biotechnology applications as well as for the pharmaceutical industry. I sites are methylated in the dna provided by bd biosciences clontech.
Pegfp c1 vector information pt30285 vector information. Is it true what with the above reagentsmaterials, i could only get transiently transfectant, but not a stable line. A daily dose of 50 lg of m dna in te 10 mm trishcl ph 7. Since the pegfp vector carries the egfp gene, any expressed fusion proteins from the pegfp plasmid will contain both the target protein and the egfp protein. To construct eukaryotic expression vector containing b71gfp geneand study its expression in osteosarcoma cell line lm8. The expression vectors pegfp c1 a and ps65t c1 b were transiently transfected into chok1 cells using a liposome transfection reagent clonfectin. Restriction map and multiple cloning site mcs of pegfpn1 vector.
Site directed mutagenesis was carried out on pegfp c1 hrpt2 9 to convert basic amino acids within each signal to neutral amino acids. All restriction sites shown dnaprovided bdbiosciences clontech. A repository of over 200,000 plasmids including protein structure initiative protein expression plasmids and vectors, over 75,000 human plasmids, and whole genome collections from many organisms. Construction of recombinant pegfpn1hper2 plasmid and its. Vector for fusing egfp to the cterminus of a partner protein. A recombinant pegfp c1 bmp2 vector was successfully constructed and overexpression of bmp2 regulated the activities of the downstream molecules of the rho gtpase signaling pathway, which might contribute to the enhancement of cell migration. Optimized codon usage and chromophore mutations provide. I am having some difficulty cloning a 1kb insert into the pegfp vector kan resistant. The xba i site is methylated in the dna provided by clontech. After being confirmed by xhoi and bamhi digestion analyses and dna sequencing, the recombinant pegfp c1 bmp2 plasmid was transfected into cos7 cells. To study the effects of bmp2 on cell migration and to explore further the molecular mechanism, a eukaryotic expression vector pegfpc1 bmp2 plasmid was constructed and transfected into cos7 cells by liposomes. Expression of two n1 clones with single amino acid. Promoters should be cloned into the pegfp 1 mcs upstream from the egfp coding sequences.