All luciferase reporter plasmids were constructed using a pgl3basic vector promega, madison, wi and all retained the mcsdre as determined by sequence analysis retrogen inc. Insert transferred from pgem9zf vector to pgl3basic vector. The reporter vectors are available in four options, depending on your needs. Downregulation of cjun expression by transcription factor. In this unit we describe how to write down vectors, how to add and subtract them, and how to use them in geometry. Isolation and cloning of human nqo1 promoter in pgl3 basic.
The luciferase reporter vectors pgl3 basic, pgl3 control, and pgl3 promoter were from promega corp. While investigating vasoactive intestinal peptide response elements in the promoter of the prolactin gene, we found that primary pituitary cells from turkey hens highly expressed endogenous. For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis see below. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression, with a modified coding region for. Promega corporation spend more, get more on promega products. The pgl3 basic luciferase reporter vector into which the cjun promoter was cloned served as a vectoralone control.
The insert was excised using the enzymes kpn i and xho i and cloned into the homologous sites of the pgl3 basic. The pgl3bcl2 promotera construct was made by digesting the pgl3bcl2 promoterfull plasmid with saci. Lipofectamine 2000, cells were transfected with control vector dna, pgl3 basic, or pgl3 stat1 promoter, in combination with either pcdna3 or gata1 expression vector pcdna3 gata1. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. Normalization of transfection efficiency using the. Single nucleotide polymorphism and cell typedependent gene. Single nucleotide polymorphism and cell typedependent. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Regulation of expression of mlaa34 gene through transcriptional. Pdf the ovalbumin gene ov gene is responsive to estrogen.
Isolation and cloning of human nqo1 promoter in pgl3 basic vector asma chinigarzadeh, razauden zulkifli, iman yaze and reyhaneh rahnamai tajadod. Downregulation of cjun expression by transcription factor c. 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 backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors. The control vector contains the sv40 promoter and enhancer region and can be used as a normalization control for expression experiments. The pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. The pgl3 basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Four firefly luciferase reporter vectors with a modified reporter gene and vector backbone. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression. All luciferase reporter plasmids were constructed using a pgl3 basic vector promega, madison, wi and all retained the mcsdre as determined by sequence analysis retrogen inc. Values are the mean sd obtained from three independent experiments. This vector is useful in the study of functional promoter elements to regulate gene expression because transcription of the luciferase gene is maintained at.
A pgl3basic vektor restrikcios emesztese es gelelektroforezise. The redesigned backbone of the pcat3 reporter vectors is similar to the pgl3 luciferase vectors with the exception of a chimeric intron located 5. Comparison of the responsiveness of the pgl3 and pgl4. Functional analysis of promoter cpg methylation using a cpg. Restriction digestion and gel electrophoresis of the pgl3basic vector. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak.
For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis. The flavonoid quercetin induces ap1 activation in frtl5. E1751 pdf 41 kb english united states dont miss out. The pgl3 vector region from the start of the reporter gene the nco i restriction site to the bacterial origin of replication sequence was also redesigned. A dualluciferase reporter assay was used to normalize for. G cgatc tcgag oh c oh t4 dna ligase tat gcatcactag taagc tttgc tctag a insert gaatt cgtcg acgag ctc nsi i spe i hind iii xba i ecor i sal i sac i xba i xho i nhe i sal i e with e in the. Generation of a novel hek293 luciferase reporter cell line. Xba i or spe i is compatible with the nhe i site of the pgl3basic vector. Ligation of vegf into pgl3 basic the digested pgl3 basic vector was treated with calf. A dioxin response element in the multiple cloning site of the. Introduction to vectors mctyintrovector20091 a vector is a quantity that has both a magnitude or size and a direction. Index terms nqo1, promoter, transcription factor, malaria, polyphenols, pgl3 vector, li. It contains a zeocin resistance gene and the r6k origin of replication. Expression was confirmed by the depositor with culturebased luminescent repoter assay.
This vector is useful in the study of functional promoter elements to regulate gene expression because transcription of the luciferase gene is maintained at a higher level by using the sv40 enhancer. In each transfection, 350 ng of each constructs and 50 ng of. A fragment of the ctsd promoter including two eboxes was cloned into pgl3basic vector and transfected into tet21n mycn on, tet21n mycn off, mycnamplified imr575, mycn singlecopy shep and sy5y cells. Promega pgl3 luciferase reporter vectors pgl3 basic vector. This may be particularly true for cos cells, which contain the sv40 large t antigen that promotes replication from the sv40 origin found in the promoter of the pgl3 promoter and pgl3 control vectors. Cells were transfected with 1 g promoterless vector pgl3basic and prl null or 5 ng vector prlcmv containing a promoter.
The psv0cat vector was obtained from the psv2cat vector by removing the sv40 simian virus 40 early promoter sequences 17. Double digestion of vegf and pgl3 basic vector, using mlu i and bgl ii the gel purified nested pcr products which were believed to be vegf promoters the insert and also pgl3 basic vector were digested by using restriction enzymes mlu i and bgl ii. Transfection is a useful technique for elucidation of molecular aspects of ion channel function. The pgl3 enhancer vector contains an sv40 enhancer downstream of the luciferase gene and the polya signal. Generation of a novel hek293 luciferase reporter cell line by. To clone the p61basal construct, pcr was performed using the p612390f and lucrp primers, and the amplicon was cloned into the pcr topo vector. The basic vector has the reporter gene and restriction enzymes sites to clone in a promoter of interest. Blackwell publishing asiafunctional roles of fli1, a. Cathepsin d protects human neuroblastoma cells from. The promoter vector is suitable for cloning and testing putative enhancer regions. Isolation and cloning of human nqo1 promoter in pgl3 basic vector. These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. Okada m 2015 single nucleotide polymorphism and cell typedependent gene expression of neuropeptide y2 receptor integr mol med, 2015 doi.
Jun 30, 2009 pgl3basic vector 4818 base pairs a plasmid vector. This vector is typically used to study upstram or downstream enhancer elements, but not promoters as there is already a promoter, tata box and other elements to run basic transcription. The enhancer vector contains the sv40 enhancer region for putative promoters that may need an additional regulator region. Basic in that it contains a sv40 enhancer element downstream of the luciferase. T84 cells have been successfully transfected with an antisense regulator of cftr. Name vector type resistance marker bacterial resistance source sequence available. The saci fragment was subcloned into the pgl3 basic vector digested with saci. Malinowska, in physiology of the gastrointestinal tract fifth edition, 2012. A dioxin response element in the multiple cloning site of. These results confirm that the pgl3 basic vector and cmv promoter both contain sequences that are transactivated by glucocorticoid treatment. A plant 35s camv promoter induces longterm expression of. Luciferase activity was measured 24 h after transfection and is reported.
The specific transcriptional characteristics of the pgl3 vectors will vary for different cell types. The pgl3control vector with the sv40 promoter upstream of the luc gene pgl3sv40 and the promoterless pgl3basic vector with the luc gene. This is a free resource for the scientific community that is compiled by addgene. The pgl3 promoter vector contains the sv40 promoter but not the enhancer. Promoter region sequence of generated vectors was confirmed by restriction digestion. Oviduct cells were transfected with pgl3 luciferase reporter plasmids and cultured alone or in the presence of 100 nm e 2, 1 m c, or e 2 and c.
Comparison of the responsiveness of the pgl3 and pgl4 luciferase. Pdf comparison of the responsiveness of the pgl3 and pgl4. Cells were transfected with 1 g promoterless vector pgl3basic and. Isolation and cloning of human vegf promoter region in pgl3. Both of these properties must be given in order to specify a vector completely. Overview protocols specifications resources the pcat3 reporter vectors provide a basis for the quantitative analysis of factors that may regulate mammalian gene expression. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to. Isolation and cloning of human vegf promoter region in.
The pgl3basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for diagnostic tools and reliable delivery of all products as quickly as possible. Mammalian 0 bacterial 0 yeast 0 worm 0 insect 0 plant 0 mouse 0 lentiviral 0 retroviral 0 adenoviral 0 aav 0 rnai 0 crelox 0. The vector, including its multiple cloning site mcs, is completely free of cpg dinucleotides.
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