Expression was confirmed by the depositor with culturebased luminescent repoter assay. Single nucleotide polymorphism and cell typedependent gene. 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. Overview protocols specifications resources the pcat3 reporter vectors provide a basis for the quantitative analysis of factors that may regulate mammalian gene expression. Isolation and cloning of human nqo1 promoter in pgl3 basic vector by razauden mohamed zulkifli, asma chinigarzadeh, iman yaze and reyhaneh rahnamai tajadod download pdf 358 kb. Luciferase activity was measured 24 h after transfection and is reported. Single nucleotide polymorphism and cell typedependent. A dualluciferase reporter assay was used to normalize for. The pgl3 enhancer vector contains an sv40 enhancer downstream of the luciferase gene and the polya signal. The control vector contains the sv40 promoter and enhancer region and can be used as a normalization control for expression experiments. Generation of a novel hek293 luciferase reporter cell line by. 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.
Basic in that it contains a sv40 enhancer element downstream of the luciferase. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. Generation of a novel hek293 luciferase reporter cell line. A plant 35s camv promoter induces longterm expression of.
A dioxin response element in the multiple cloning site of the. The pgl3 basic luciferase reporter vector into which the cjun promoter was cloned served as a vectoralone control. 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. 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. Pdf comparison of the responsiveness of the pgl3 and pgl4. These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. The pgl3 basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Restriction digestion and gel electrophoresis of the pgl3basic vector. 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. Promega corporation spend more, get more on promega products.
T84 cells have been successfully transfected with an antisense regulator of cftr. 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. Transfection is a useful technique for elucidation of molecular aspects of ion channel function. Downregulation of cjun expression by transcription factor c. In each transfection, 350 ng of each constructs and 50 ng of. Index terms nqo1, promoter, transcription factor, malaria, polyphenols, pgl3 vector, li. The basic vector has the reporter gene and restriction enzymes sites to clone in a promoter of interest. Functional analysis of promoter cpg methylation using a cpg. Isolation and cloning of human nqo1 promoter in pgl3 basic vector asma chinigarzadeh, razauden zulkifli, iman yaze and reyhaneh rahnamai tajadod. The insert was excised using the enzymes kpn i and xho i and cloned into the homologous sites of the pgl3 basic. 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. A pgl3basic vektor restrikcios emesztese es gelelektroforezise.
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. Isolation and cloning of human nqo1 promoter in pgl3 basic. The reporter vectors are available in four options, depending on your needs. The pgl3bcl2 promotera construct was made by digesting the pgl3bcl2 promoterfull plasmid with saci. 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. This vector is useful in the study of functional promoter elements to regulate gene expression because transcription of the luciferase gene is maintained at. Cathepsin d protects human neuroblastoma cells from. Downregulation of cjun expression by transcription factor.
Xba i or spe i is compatible with the nhe i site of the pgl3basic vector. For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis. 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. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression. The vector, including its multiple cloning site mcs, is completely free of cpg dinucleotides. Okada m 2015 single nucleotide polymorphism and cell typedependent gene expression of neuropeptide y2 receptor integr mol med, 2015 doi. Normalization of transfection efficiency using the. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. Jun 30, 2009 pgl3basic vector 4818 base pairs a plasmid vector. The pgl3basic 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. Promega pgl3 luciferase reporter vectors pgl3 basic vector. This is a free resource for the scientific community that is compiled by addgene. The saci fragment was subcloned into the pgl3 basic vector digested with saci. Isolation and cloning of human vegf promoter region in. The transformation with pgl3 basic control vector works pretty well in all e. 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.
The flavonoid quercetin induces ap1 activation in frtl5. 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. Cells were transfected with 1 g promoterless vector pgl3basic and. E1751 pdf 41 kb english united states dont miss out. The pgl3control vector with the sv40 promoter upstream of the luc gene pgl3sv40 and the promoterless pgl3basic vector with the luc gene. Both of these properties must be given in order to specify a vector completely. It contains a zeocin resistance gene and the r6k origin of replication. Pdf the ovalbumin gene ov gene is responsive to estrogen. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression, with a modified coding region for. The specific transcriptional characteristics of the pgl3 vectors will vary for different cell types. The enhancer vector contains the sv40 enhancer region for putative promoters that may need an additional regulator region.
Isolation and cloning of human nqo1 promoter in pgl3 basic vector. The psv0cat vector was obtained from the psv2cat vector by removing the sv40 simian virus 40 early promoter sequences 17. For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis see below. Ligation of vegf into pgl3 basic the digested pgl3 basic vector was treated with calf. 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.
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. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors. Comparison of the responsiveness of the pgl3 and pgl4. 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. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Isolation and cloning of human vegf promoter region in pgl3. Regulation of expression of mlaa34 gene through transcriptional. The promoter vector is suitable for cloning and testing putative enhancer regions. The luciferase reporter vectors pgl3 basic, pgl3 control, and pgl3 promoter were from promega corp. Comparison of the responsiveness of the pgl3 and pgl4 luciferase. A dioxin response element in the multiple cloning site of. 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 pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression.
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