As with the pgl3 vectors, the pcat3 vectors contain a different polyadenylation site located 3. This website uses cookies to ensure you get the best experience. In particular, promega has developed a firefly luciferase reporter system to study the regulation of gene expression in mammalian cell culture. Promega pgl3 luciferase reporter vectors four firefly luciferase reporter vectors.
In the present study, we compared the role of genetic variations of the tlr4 gene in susceptibility to copd and ptb and illuminated the underlying molecular mechanism of functional singlenucleotide polymorphisms snps. Cryptic enhancer elements in luciferase reporter vectors respond. Human maf1 negatively regulates rna polymerase iii transcription via the tfiib family members brf1 and brf2. Janet rollins 1, ingrid veras 1, stephanie cabarcas 1, ian willis 2, laura schramm 1. This plasmid is useful in monitoring transfection efficiency, in general, and is a convenient internal standard for promoter and enhancer activities expressed by pgl3 recombinants. P oct 22, 2019 chronic obstructive pulmonary disease copd and pulmonary tuberculosis ptb share a number of common risk factors, including innate immunityrelated genetic factors. This file type can store an enormous amount of graphics information and is editable in adobe illustrator. A dioxin response element in the multiple cloning site of. Dna fragments containing putative enhancer elements can be inserted.
Vectors are supplied with a glycerol stock of bacterial strain jm109 cells. Nov 03, 2009 the luciferase assay is one of the most wellknown and convenient reporter assays. Cells were also transfected with the empty pgl3basic vector or pgl3control vector sv40 pgl3 as negative or positive controls, respectively. A dioxin response element in the multiple cloning site of the pgl3 luciferase reporter influences transcriptional activity 1. Evaluating the microrna targeting sites by luciferase.
To determine whether this sequence confers promoter activity, several cell lines including tc7 were transiently transfected with the pgl3bco1022 reporter. Shop online for a wide selection of promega pgl3 luciferase reporter vectors four firefly luciferase reporter vectors. Generation of the spdef, foxa3, and agr2 expression plasmids. The redesigned backbone increases expression of the reporter gene, improves in vivo vector stability and provides greater flexibility in performing manipulations. Iman yaze, razauden zulkifli, asma chinigarzadeh, reyhaneh rahnamai tajadod isolation and cloning of human vegf promoter region in pgl3 basic vector published at. It came out that i have colonies for the control pgl3 vector sample, but not in my ligated vectors samples.
Isolation and cloning of human nqo1 promoter in pgl3 basic vector asma chinigarzadeh, razauden zulkifli, iman yaze and reyhaneh rahnamai tajadod. The pcat3 reporter vectors provide a basis for the quantitative analysis of factors that may regulate mammalian gene expression. This is a free resource for the scientific community that is compiled by addgene. The pgl3 enhancer vector contains an sv40 enhancer downstream of the luciferase gene and the polya signal. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. International journal of scientific and research publications ijsrp, volume 2, issue 8, august 2012 edition. Promoter region sequence of generated vectors was confirmed by restriction digestion. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide.
Regulation of expression of mlaa34 gene through transcriptional. The prlsv40 vector a is intended for use as an internal control reporter vector and may be used in combination with any experimental reporter vector to cotransfect mammalian cells. The pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. Restriction digestion of pgl3 basic vector and separation of dna. Rna polymerase iii transcription can be measured in vivo using a rna pol iii dual luciferase assay. The pgl3 luciferase reporter vectors offer different combinations of the sv40. The pgl3 luciferase reporter vectorsa,b provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. That means theres prolly something wrong with my ligation pgl3 vectors, or my insert.
The pgl3 reporter vectors contain a modified firefly. In this chapter, we use pgl3 control vector promega, one of the most commonly used luciferase reporter vectors. For use with application quantitative analysis of factors that potentially regulate mammalian gene expression. For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis see below. Restriction digestion and gel electrophoresis of the pgl3basic vector. To confirm the transcriptional origin of the regulation of mir155 under tnf. A dioxin response element in the multiple cloning site of the.
Ligation of vegf into pgl3 basic the digested pgl3 basic vector was treated with calf. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. This vector is useful in the study of functional promoter elements to regulate gene expression because transcription of the luciferase gene is maintained at. The pgl4 vector backbone to reduce the risk of anomalous expression and increase the reliability of reporter gene expression, the pgl3 vector region upstream of the reporter gene was reengineered to create the pgl4 vectors. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. The pgl3basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences.
Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. The pgl3 basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. The reporter vectors are available in four options, depending on your needs. The enhancer vector contains the sv40 enhancer region for putative promoters that may need an additional regulator region. The prlsv40 vector contains the sv40 enhancer and early promoter elements to provide highlevel expression of renilla luciferase in cotransfected mammalian cells. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. 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. Atf3 inhibits the tumorigenesis and progression of. Isolation and cloning of human vegf promoter region in. 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.
The pgl3 luciferase vectors were obtained from promega, and the. Set up digests as described above, as if you were going to ligate the plasmid to an insert ie. The pgl3 luciferase reporter vectors a provide a basis for the quantitative analysis of factors that potentially regulate. This aids in the verification of functional promoter elements because the presence. A schematic diagram of a pgl4 vector is shown in figure 1. These factors may be cisacting, such as promoters and enhancers, or.
A transient transfection of hela cells with increasing concentrations of pgl3u6 100ng, 200ng, 300ng, pgl3vai 100ng, 200ng, 300ng or empty pgl3 vector 300ng. The insert region for my vector is only 500 something bp. The pgl3promoter vector contains an sv40 promoter upstream of the luciferase gene. The target primer sequences are listed in additional file 1. Obesityassociated inflammation induces microrna155. Isolation and cloning of human vegf promoter region in pgl3. The basic vector has the reporter gene and restriction enzymes sites to clone in a promoter of interest. 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.
Isolation and cloning of human vegf promoter region in pgl3 basic vector iman yaze, razauden zulkifli, asma chinigarzadeh and reyhaneh rahnamai tajadod faculty of bioscience and bioengineering, 810 universiti technologi malaysia abstract worldwide mortality and morbidity from infectious. Wilcoxon test p pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. International journal of scientific and research publications ijsrp. Im having a hard time cloning 2 fragments 2,2kb and 2,3kb into pgl3 promoter vector 5lb, for some time now. 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. In this chapter, we use pgl3control vector promega, one of the most commonly used luciferase reporter vectors.
Numerous configurations of pgl4 vectors are available. The pgl4 luciferase reporter vectors are the next generation of reporter gene vectors optimized for more efficient expression in mammalian cells. The specific transcriptional characteristics of the pgl3 vectors will vary for different cell types. By continuing to use this site, you agree to the use of cookies. Nitric oxide reduces slc29a1 promoter activity and. To convert pdf to vector format, it is necessary to convert a pdf to bitmap image firstly and then you can easily convert the images to vectors. Easy way on how to convert pdf to vector files iskysoft. To determine whether this sequence confers promoter activity, several cell lines including tc7 were transiently transfected with the pgl3bco1022 reporter data not shown. These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. 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. Human maf1 negatively regulates rna polymerase iii. I amplify interest fragment by pcr, then i make a double digestion with mluibglii restriction enzymes of insert and plasmide. Department of biochemistry, albert einstein college of medicine.
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