Aug 25, 2011 the iresegfp fusion was constructed by the method of splicing by overlapping extension soepcr 44, 45 using the templates pegfpc1 clontech, mountain view, ca, usa and the primers 5. Also needed for this protocol are egfpc1 clontech, psub201 18, repcap. Interaction of human biliverdin reductase with aktprotein. The gfppin antisense vector was obtained by cloning the pin1 pcr fragment with ecori, bamhi into the ecori, bamhi sites of pegfpc1 clontech. Nov 21, 2010 yo biofreakers, today in the ggs live section we will learn how to virtually design and generate a fusion protein.
Human cytomegalovirus rl protein interacts with host nudt14. The jembrana disease virus jdv, a lentivirus, is the etiologic agent of. 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. The cells were pulsed in the presence of the pegfpc1 plasmid dna with the following parameters. Construction of a eukaryotic expression vector pegfpc1. Generation and characterization of the first immortalized. In cotransfection experiments, a plasmid ratio of 1. Cortactintdtomato ctnntdtom was generated by cloning cttn coding sequence from cttnegfp into ptdtomaton1 clontech. Mountain view, ca, usa, pdsred2c1 clontech laboratories, andor pet28a novagen, madison, wi, usa by standard methods. 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.
Using the pegfpc1 vector with lipofectamine2000 on 293t cells, you can easily produce stably transfected cells. For gfpadar1 p110, the adar1 p110 sequence amino acids aa 2961226. Restriction map and multiple cloning site mcs of pacgfp1c1. To fulfill its function, rev shuttles between the cytoplasm and the nucleus. Direct binding assay lysates in 50 mm trishcl, ph 7. The lentiviral rev protein, which is a regulatory protein essential for virus replication, has been first studied in the human immunodeficiency virus type 1 hiv1. Marco marcellis lab contains the insert androgen receptor ar and is published in prostate. Biosciences, little chalfont, uk and pegfpc1 clontech bd biosciences, oxford, uk.
Producing a mammalian gfp expression vector containing. Actinmcherry was generated by cloning actin coding sequence from actinegfp into pmcherryc1 clontech. Hcmv and tet promoters for inducible gene expression in rat. Expression of transfected pegfpaldoa panels a and e, immunostaining for dsrna panels b and f, and dapistained nuclei panels c and g are shown. Fulllength bovine aux1 910 residues was amplified by pcr from a fulllength cdna clone and inserted into pegfpc1 clontech, resulting in pegfpaux1.
Phosphorylationdependent proline isomerization catalyzed. Sequences flanking egfp have been converted to a kozak consensus translation initiation site 6 to further increase the translation efficiency in eukaryotic cells. The mcs in pegfpn1 is between the immediate early promoter of cmv p. Furthermore the alpaca, as well as the rest of the camelids, possesses the peculiarity of producing singlechain antibodies from which nanobodies can be generated. Lc3a cdna was transferred into pegfpc1 clontech toproducegfplc3. This is a free resource for the scientific community that is compiled by addgene.
The ph domains of the akts were obtained by amplification of aa 1250 of each isoform and subsequent cloning into the pegfpc1,pdsred2c1,andpet28a vectors. Nanobodies, due to their structural simplicity and reduced size, are. The xba i and bcl i sites are methylated in the dna provided by bd biosciences clontech. N and c are, as you thought the nterminal and cterminal tags, often it is a bit counterintuitive for these though, for example in the pegfpc1 plasmid, if you clone into this, the gfp will be nterminal to the insert i. You are correct in that the neomycin gene in pegfpc1 will allow you to stablilse using g418. Fulllength hmis12 was pcr amplified from potb7hmis12 and ligated into pcr3 containing an nterminal vsvtag to obtain vsvhmis12. Ecor i, and cloned into xhoi and ecor i site of pegfpc1 clontech. The plasmids encoding the cenpb and mis12incenp fusion cdnas were generated as follows. At 75% confluence, the cells were transfected with either 4 g pegfpc1rl, 4 g pdsredc1nudt14, or a mixture of 2 g pegfpc1rl and 2 g pdsredc1nudt14 using lipofectamine 2000 invitrogen. I also have pegfp plasmid that i know very little about. Uncoating atpase hsc70 is recruited by invariant chain and. N and c are, as you thought the nterminal and cterminal tags, often it is a bit counterintuitive for these though, for example in the pegfp c1 plasmid, if you clone into this, the gfp will be nterminal to the insert i.
Vector for fusing egfp to the nterminus of a partner protein. Gst fusion proteins of pctaire1, and mutants thereof, were expressed in bl21de3 e. Saliapa1 pegfp flpld1, salibamh1 pegfp nfpld1 and ecor1apa1 pegfp cfpld1 sites in polylinker regions of pegfp c1 clontech. Transfection and selection data related to pegfp c3 in cellculture. Learn about the latest plasmid technologies and research tools. Lc3g120a andlc3k51a cdnas were prepared with quikchange ii sitedirected mutagenesis kit stratagene. Restriction map and multiple cloning site mcs of pegfpc2. Using the pegfp c1 vector with lipofectamine2000 on 293t cells, you can easily produce stably transfected cells. At 48 h posttransfection, the cells were subjected to dapi. I sites are methylated in the dna provided by bd biosciences clontech. Mountain view, ca, usa, pdsred2 c1 clontech laboratories, andor pet28a novagen, madison, wi, usa by standard methods. Enhancement of green fluorescent protein expression in adeno.
Download fulltext pdf rgs4 and rgs2 bind coatomer and inhibit copi association with golgi membranes and intracellular transport article pdf available in molecular biology of the cell 119. Mammalian expression vector, adds cterminal gfp tag. The expression vector pegfpc1 alone was used as a control c and d. For mammalian expression, the respective cdnas were cloned in frame to gfp into pegfpc1 clontech, palo alto, ca. The iresegfp fusion was constructed by the method of splicing by overlapping extension soepcr 44, 45 using the templates pegfpc1 clontech, mountain view, ca, usa and the primers 5. The mcs in pegfp c1 is between the egfp coding sequences and the sv40 poly a. Hcmv and tet promoters for inducible gene expression in rat neurons in vitro and in vivo. A burst of auxilin recruitment determines the onset of. Psa promoterdriven luciferase reporter vector ppsa6.
Construction of a eukaryotic expression vector pegfpc1bmp2. The cells were harvested for coimmunoprecipitation and western blot analysis. Our mission is to develop highquality innovative tools and services to accelerate discovery. Takara bio provides kits, reagents, and services that help researchers explore questions about gene discovery, regulation, and function. Electroporated cells were transferred to new 25 cm 2 flask and fed with complete medium. Pdf inhibition of aldolase a blocks biogenesis of atp. Sciences for bacterial expression as a protein fused to glutathione s. The gcamp6f gene was amplified from pgpcmvgcamp6f addgene, plasmid 40755 and inserted into pegfpc1 clontech. With regards to the correct concentration, may i suggest you perform a g418 titration curve. Correlation of 4pi and electron microscopy to study.
Brighter reporter genes from multimerized fluorescent proteins. Our protocol is to transfect when cells are 50% confluent and i combine 78 ul pei 1mgml with 194ul. This project is supported bytokuewhich specializes in manufacturing ultrapure antibiotics for a broad spectrum of biotechnology applications as well as for the pharmaceutical industry. Asscs from a subconfluent 25 cm 2 flask were also electroporated equibio apparatus. Digital collection of empty plasmid backbones from publications and commercially available sources. Expression of zac1 and the house keeping gene gapdh were detected by reverse transcriptase polymerase chain reaction rt. If you wish to digest the vector with these enzymes, you will need to transform the vector into a dam host and make fresh dna. Interestingly, the migration of cells transfected with pegfp c1 bmp2 plasmid was significantly increased at 36 and 48 h compared with the control group and the pegfp c1 group p pegfp c1 ar3 wednesday, may 12, 2010 03.
Feb 28, 2009 after transformation of li top10f strain cells with pegfp n1 vector and pcdna3. The main function of rev is to mediate the nuclear exportation of viral rnas. Interaction of hepatitis c virus f protein with prefoldin. The cterminal jdomain of aux1 was removed by using an internal bamhi site to create a truncation after m812 pegfpaux1.
Suppression of the novel er protein maxer by mutant. The xba i site is methylated in the dna provided by clontech. Hamamatsu imageem, and an lmm5 laser merge module spectral applied research, controlled by ip lab software bd biosciences. Interestingly, the migration of cells transfected with pegfpc1bmp2 plasmid was significantly increased at 36 and 48 h compared with the control group and the pegfpc1 group p clontech laboratories, inc. Anillin phosphorylation controls timely membrane association.
Among this group, hpv16 is the most common type, being found in about fifty percent of invasive cancers worldwide. Genes cloned into the mcs will be expressed as fusions to the cterminus of egfp if they are in the same reading frame as egfp and there are no intervening stop codons. Xba i and bcl i sites are methylated in the dna provided by clontech laboratories, inc. The rat triadin 1 and junctin genes were cloned and integrated into pegfpc1gcamp6f with gcamp6f at the n terminus. You are correct in that the neomycin gene in pegfp c1 will allow you to stablilse using g418. Cn101974554a cn 201010291680 cn201010291680a cn101974554a cn 101974554 a cn101974554 a cn 101974554a cn 201010291680 cn201010291680 cn 201010291680 cn 201010291680 a cn201010291680 a cn 201010291680a cn 101974554 a cn101974554 a cn 101974554a authority cn china prior art keywords pegfp n1m egfp n1 expression prior art date 20100926 application. Raising of alpacas as exotic livestock for wool and meat production and as companion animals is growing in importance in the united states, europe and australia. Clontech backbone size w o insert bp 4731 total vector size bp 4766. Atcc, manassas, va, usa were transfected with these constructs using lipofectamine 2000 invitrogen, carlsbad, ca, usa.
Save the supernatant and combine with the original 15 ml lysate from step 17. Optypuls with 10 g of pegfpc1 dna in dmem high with 10% fbs. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Hcmv and tet promoters for inducible gene expression in. Fulllength pes1 amplified from human cdna by pcr was cloned into pegfpc1 clontech, mountain view, usa and pgex4t1 ge healthcare, piscataway, usa generating gfppes1 and gstpes1, respectively. To verify the interaction between hcv f protein and pfd2, two plasmids, pegfphcvf and pcdnapfdn2, were constructed to express the egfphcvf and pfd2v5 fusion proteins fig.
Restriction map and multiple cloning site mcs of pegfpn1 vector. Hek293 cells were observed 24 h after transfection using a leica inverted fluorescent microscope under normal light b and d and with leica fluorescein isothiocyanate filter sets a and c. Transfection and selection data related to pegfp c1 in cellculture. The primers for establishment of various pld1 constructs are. Human papillomavirus hpv is a ubiquitous sexually transmitted dna virus. The expression vector pegfpc1 clontech was used to generate fusion protein constructs with gfp at the n terminus of ar and the nar mutant for convenient visualization using. The iresegfp fusion was constructed by the method of splicing by overlapping extension soepcr 44, 45 using the templates pegfp c1 clontech, mountain view, ca, usa and the primers 5.
If you wish to digest the vector with this enzyme, you will need to transform the vector into a dam host and make fresh dna. Receive the latest news, hot plasmids, discounts and more. Mountain view, ca 94043 technical support us email. Drebrin a appears in neurons coincident with synaptogenesis and accumulates at post. Inhibition of androgen receptor nuclear localization and. Cos1 cells were transfected by using a deaedextran procedure. C1 vector using the bglii and hindiii restriction sites inserted in the forward and reverse primers, respectively.
Restriction map and multiple cloning site mcs of pegfpc1. In all cloning experiments, the vector was cut by the appropriate restriction enzyme and run on a 1% agarose gel. Hek293 cells were transfected with different combination of these two plasmids and pegfpc1 encoding only egfp fig. The mcs in pacgfp1c1 is downstream of the acgfp1 coding region, allowing the contruction of a cterminal fusion protein with the acgfp1 when genes are cloned in the same reading frame as acgfp1 and there are no intervening stop codons. Fulllength b23 cloned from human cdna was inserted into pet28a novagen, madison, usa to encode 6. If you wish to digest the vector with this enzyme, you will need to transform the vector into a dam and make fresh dna. Proteins that contain a functional zdnabinding domain. Fulllength human anillin cdna isoform 2 accession number bc070066, open biosystems was cloned into pegfpc1 clontech.
The ph domains of the akts were obtained by amplification of aa 1250 of each isoform and subsequent cloning into the pegfp c1,pdsred2 c1,andpet28a vectors. Phosphorylationdependent proline isomerization catalyzed by. This isoform was used previously to analyze anillin domain functions and encodes a protein with a 37 residue gap between the actinbinding and rhobinding domains, compared with the longest isoform. Zac1 regulates astroglial differentiation of neural stem.
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