First quantify the plasmid by gel comparison, not nanodrop. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression. All sirnas, the mir520b inhibitor, and the control inhibitor were purchased from. The enhancer vector contains the sv40 enhancer region for putative promoters that may need an additional regulator region. Human maf1 negatively regulates rna polymerase iii. Duelluciferase reporter assay the wide type and the mutation of the promoter segment sequences of integrin. The pgl3 promoter vector contains the sv40 promoter but not the enhancer. 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. The pgl3 luciferase reporter vectors offer different combinations of the sv40 early promoter and enhancer sequences to provide flexible experimental strategies and user convenience. The system comprises complex carbohydrate structures that are biosynthesized by the a and b transferases encoded by the a and b genes, respectively. We used the dualluciferase reporter assay system promega to measure the activity of the firefly luciferase gene and the renilla luciferase gene. Dna sequencing confirmed the identity of all constructs. 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 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 promoter vector is suitable for cloning and testing putative enhancer regions. Three pgl4 reporter vectors that express luciferase proteins with different stabilities for investigating gene expression control regions. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources.
The pgl3basic vector without an insert sequence was used as a negative control. International journal of scientific and research publications ijsrp. To determine whether this sequence confers promoter activity, several cell lines including tc7 were transiently transfected with the pgl3bco1022 reporter data not shown. Taken together these data suggest that geneinternal and external rna pol iii. Supplementary information for kewei zheng, yide he, hong. Expression was confirmed by the depositor with culturebased luminescent repoter assay. The pcpglbasic vector was chosen instead of the conventional pgl3basic vector due to a cpg dinucleotidefree backbone, which allows observation of methylation effects on the inserted promoter fragments rather than detecting artificial effects of reporter gene i. 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. The numbers in the parenthesis indicate the distances between the 3 end of the gcores and the 5 end of the promoters. Pshai quickly loses activity at 37c, but can be used at 25c for long incubations. Stay notified of promega events, products and news. Luciferase activity was detected 48 h after transfection.
Sitedirected mutagenesis was performed using a quickchange sitedirected mutagenesis kit stratagene. Hindiii site in the pgl3basic vector to generate the pgl3il6 construct. Gcore sequence, one each plasmid, was placed on the template strand. Expression of abo bloodgroup genes is dependent upon an.
Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The pgl3 enhancer vector contains an sv40 enhancer downstream of the luciferase gene and the polya signal. The abo blood group system is of great importance in blood transfusion and organ transplantation. Isolation and cloning of human vegf promoter region in. A dioxin response element in the multiple cloning site of. Promega pgl3 luciferase reporter vectors pgl3 basic vector. Glial cell linederived neurotrophic factor gdnf is a potent survival factor for many cell types, and its expression is widespread both within and outside of the nervous system. The vai gene was pcr amplified from padvantage and ligated into pgl3basic, yielding pgl3vai which was transiently transfected into hela cells and demonstrated a dose response increase in luciferase activity, as compared to the empty pgl3 vector figure 1a. The pgl3basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Vascular endothelial growth factor induces multidrug. Functional mapping of genetic elements can be achieved through nested deletions, and singlestranded dna may be prepared for mutagenesis or sequencing.
To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. The transformation with pgl3 basic control vector works pretty well in all e. Using a luciferase reporter assay, we identified the role of the 3untranslated region 3utr of. Characterization of 3untranslated region of the mouse. Luciferase transcriptional activity measured in hela cells transiently trans fected with the showed reporter constructs is reported in the histogram. Four firefly luciferase reporter vectors with a modified reporter gene and vector backbone.
Luciferase activity was detected 48h after transfection. Obesityassociated inflammation induces microrna155. E1751 pdf 41 kb english united states dont miss out. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. Functional variations of the tlr4 gene in association with. The construct or the empty pgl3basic vector was then transiently transfected into bgc823 cells, along with the control vector. For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis see.
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 order to explore the functions of vegf affecting the mrp1 promoter, we synthesized the promoter of mrp1 and cloned it into the luciferase reporter gene vector pgl3basic, named pgl3basicmrp1w. This is a free resource for the scientific community that is compiled by addgene. Ligation of vegf into pgl3 basic the digested pgl3 basic vector was treated with calf. 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. Pax2 oncogene negatively regulates the expression of the. Isolation and cloning of human nqo1 promoter in pgl3 basic vector asma chinigarzadeh, razauden zulkifli, iman yaze and reyhaneh rahnamai tajadod. Sticky ends from different acci sites may not be compatible. Iman yaze, razauden zulkifli, asma chinigarzadeh, reyhaneh rahnamai tajadod isolation and cloning of human vegf promoter region in pgl3 basic vector published at.
The chimeric intron bearing construct is also depicted. For each constructed vector, independent triplicate. The control vector contains the sv40 promoter and enhancer region and can be used as a normalization control for expression experiments. Promega corporation 2800 woods hollow road madison, wi 537115399 usa. The pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. Transcription mediated gquadruplex formation in plasmids containing a gcore. Twentyfour hours after transfection, the cells were lysed in 200.1479 778 1213 2 1156 1051 207 450 171 1042 709 1024 1286 173 738 1470 431 1320 1378 27 1314 1271 930 1418 1488 1417 1242 2 1265 9 957