The xba i site is methylated in the dna provided by clontech. Tbusa, formerly known as clontech laboratories, inc. The eukaryotic expression vector pegfpc1bmp2 was originally generated and. 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. Restriction map and multiple cloning site mcs of pacgfp1c1. The vectors pgfpmutlc1 and pegfpc1 clontech each encode the gfpmutl variant. How to obtain a stable transfectant aug022005 hi, all. Construction of a eukaryotic expression vector pegfpc1.
The duck pegfpfst vector was successfully constructed and was confirmed to. Molecular cloning and characterization of stamp2, an. The recombinant pegfp c1 bmp2 was effectively expressed in cos7 cells. Intensiometric biosensors visualize the activity of multiple small. By using gene cloning technique, eukaryotic expression vector pegfp c1 was used to construct the murine b71 recombinant plasmid pegfp c1 b7. 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. Mammalian expression vector, adds cterminal gfp tag.
Deep sequencing reveals complex spurious transcription from. Xba i and bcl i sites are methylated in the dna provided by clontech laboratories, inc. Vector for fusing egfp to the cterminus of a partner protein. Stable transfection of pegfp n1mog plasmid to utilize in multiple sclerosis gene therapy. Im trying to set up the transfection technique in my new lab. I ags cells were transiently transfected with pegfpc1 plasmid egfp, pegfpc1 containing y5. 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. Pegfp c1 vector information pt30285 vector information. The c2 domains of the class i rab11 family of interacting. I sites are methylated in the dna provided by bd biosciences clontech. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The ecori and xhoidigested segment coding for the truncated mutant rip3 n223 was inserted into ecorisalidigested pegfp c1 to yield. Expression of two n1 clones with single amino acid. The expression vectors pegfp c1 a and ps65t c1 b were transiently transfected into chok1 cells using a liposome transfection reagent clonfectin.
The results revealed that in the pegfp n1hper2 and the pegfp n1 group, large numbers of mg63 expressed gfp. 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 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. Benchling works best when using a supported browser. Construction of a eukaryotic expression vector for pegfp. The xbai site is methylated in the dna provided by clontech. Gli3 mutations in human disorders mimic drosophila cubitus. Gfprip111218was generated by digesting pegfp c1 rip11 with bamhi and religating the backbone. I know my ligated product is present via pcr and by simply running it on a gel. Am using clontech s pegfp c1 vector along with lipofectamine2000 on 293t cells.
Description pegfp c1 encodes redshiftedvariant wildtypegfp whichhas been optimized brighterfluorescence higherexpression mammaliancells. Without the addition of a functional promoter, this vector will not express. 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. 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.
The substituted nucleotides encoded the amino acid sequence gplgs. For license information, please contact a licensing representative by phone at 650. Recombinant plasmid was transfected into lm8 cells with liposome and was confirmed by. The integrity of the final pegfp c1 cyp26a1 construct was confirmed by sequence analysis. Hi all, i am new to the forums, but i have been a longtime lurker when the need arose.
Optimized codon usage and chromophore mutations provide. To construct eukaryotic expression vector containing b71gfp geneand study its expression in osteosarcoma cell line lm8. Identification and characterization of a ligandregulated. The amplified fragment was digested with bglii and xhoi and then cloned into bgliixhoi sites of pegfp c1 clontech to generate pegfp c1 rip3. The effects of the overexpressed bmp2 on the migration of cos7 cells and the underlying molecular mechanism were investigated. The vector pegfp c1 has been already utilised in gene expression in cell culture model xu et al.
After being confirmed by xhoi and bamhi digestion analyses and dna sequencing, the recombinant pegfp c1 bmp2 plasmid was transfected into cos7 cells. Expression of egfp and gfps65t in transfected chok1 cells. Construction of a eukaryotic expression vector for pegfpfst and. For other reading frames, use pegfp n1 or pegfp n2. Hindiiibamhi fragment from pegfp c1 rab11fip2 into the hindiiibamhi site of pegfp c2 clontech. Forprofit entities wishing to use this product are required to obtain a license from clontech. I am having some difficulty cloning a 1kb insert into the pegfp vector kan resistant. Restriction map and multiple cloning site mcs of pegfpc1. 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. 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. Is it true what with the above reagentsmaterials, i could only get transiently transfectant, but not a stable line. Bamhi fragment was ligated into the response plasmid, pegfp enhanced green fluorescent protein c1 clontech, palo alto, ca designated as pegfp c1 cyp26a1 plasmid. This nes ar mutant was cloned into pegfp c1 to produce pegfp nes argplgs 762. Construction of recombinant pegfpn1hper2 plasmid and its.
Engineering of a monomeric greentored photoactivatable. The green fluorescent protein gfp from aequorea victoria is a versatile reporter protein for. 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. Construction of eukaryotic expression vector containing b7. Promoters should be cloned into the pegfp 1 mcs upstream from the egfp coding sequences. Construction of a eukaryotic expression vector pegfpc1 bmp2 and its effect on cell migration by. The resulting pcr product was cloned into the same sites in the vector pegfp c1 clontech. Grb2 is a key mediator of helicobacter pylori caga protein activities.
Restriction map and multiple cloning site mcs of pegfpn1 vector. As a control vector, pcmsegfpcyp26a1 plasmid, which has separate transcription cassettes. 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. An orange fluorescent protein with a large stokes shift for singleexcitation multicolor fccs and fret imaging. The influence of bmp2 on cell migration and cofilin activity was detected by cell scratch assay and western blotting. Rafused rac1 or cdc42 were pcramplified and fused to egfpc1 clontech vector after excision of egfp at the site of nhei and bamhi. Site directed mutagenesis was carried out on pegfp c1 hrpt2 9 to convert basic amino acids within each signal to neutral amino acids. 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. The fulllength stamp2 orf from pcriitopostamp2 was fused in frame to the cterminus of gfp using the vector pegfp c1 clontech. An orange fluorescent protein with a large stokes shift.
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