Pshai quickly loses activity at 37c, but can be used at 25c for long incubations. The pgl3 luciferase reporter vectors offer different combinations of the sv40 early promoter and enhancer sequences to provide flexible experimental strategies and user convenience. To confirm the transcriptional origin of the regulation of mir155 under tnf. 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. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. The pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. Gcore sequence, one each plasmid, was placed on the template strand. Functional mapping of genetic elements can be achieved through nested deletions, and singlestranded dna may be prepared for mutagenesis or sequencing.
The system comprises complex carbohydrate structures that are biosynthesized by the a and b transferases encoded by the a and b genes, respectively. The numbers in the parenthesis indicate the distances between the 3 end of the gcores and the 5 end of the promoters. 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. For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis see. 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. Oncoprotein hbxip enhances hoxb acetylation and co.
Vascular endothelial growth factor induces multidrug. Nuclear lncrna hoxdas1 suppresses colorectal carcinoma. Isolation and cloning of human vegf promoter region in. For each constructed vector, independent triplicate. 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. Isolation and cloning of human nqo1 promoter in pgl3 basic vector asma chinigarzadeh, razauden zulkifli, iman yaze and reyhaneh rahnamai tajadod. To determine whether this sequence confers promoter activity, several cell lines including tc7 were transiently transfected with the pgl3bco1022 reporter data not shown. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression. We used the dualluciferase reporter assay system promega to measure the activity of the firefly luciferase gene and the renilla luciferase gene. The pgl3 enhancer vector contains an sv40 enhancer downstream of the luciferase gene and the polya signal.
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. The regulation of gdnf expression has been extensively investigated but is not fully understood. Supplementary information for kewei zheng, yide he, hong. 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. Human maf1 negatively regulates rna polymerase iii.
Ligation of vegf into pgl3 basic the digested pgl3 basic vector was treated with calf. Functional variations of the tlr4 gene in association with. Characterization of 3untranslated region of the mouse. The pgl3 promoter vector contains the sv40 promoter but not the enhancer.
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 pgl3basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. 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 basic vector has the reporter gene and restriction enzymes sites to clone in a promoter of interest. Expression of abo bloodgroup genes is dependent upon an. International journal of scientific and research publications ijsrp.
Promega corporation 2800 woods hollow road madison, wi 537115399 usa. Luciferase transcriptional activity measured in hela cells transiently trans fected with the showed reporter constructs is reported in the histogram. Taken together these data suggest that geneinternal and external rna pol iii. The transformation with pgl3 basic control vector works pretty well in all e. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. E1751 pdf 41 kb english united states dont miss out. Promega pgl3 luciferase reporter vectors pgl3 basic vector. This is a free resource for the scientific community that is compiled by addgene.
Obesityassociated inflammation induces microrna155. Iman yaze, razauden zulkifli, asma chinigarzadeh, reyhaneh rahnamai tajadod isolation and cloning of human vegf promoter region in pgl3 basic vector published at. The enhancer vector contains the sv40 enhancer region for putative promoters that may need an additional regulator region. Transcription mediated gquadruplex formation in plasmids containing a gcore.
Hindiii site in the pgl3basic vector to generate the pgl3il6 construct. A dioxin response element in the multiple cloning site of. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The construct or the empty pgl3basic vector was then transiently transfected into bgc823 cells, along with the control vector. Sitedirected mutagenesis was performed using a quickchange sitedirected mutagenesis kit stratagene. The abo blood group system is of great importance in blood transfusion and organ transplantation. Expression was confirmed by the depositor with culturebased luminescent repoter assay.
The promoter vector is suitable for cloning and testing putative enhancer regions. The chimeric intron bearing construct is also depicted. First quantify the plasmid by gel comparison, not nanodrop. Using a luciferase reporter assay, we identified the role of the 3untranslated region 3utr of. Twentyfour hours after transfection, the cells were lysed in 200. Three pgl4 reporter vectors that express luciferase proteins with different stabilities for investigating gene expression control regions. The boundaries of the ubc promoter regions intronbearing and intronless cloned into the pgl3basic vector are indicated. Luciferase activity was detected 48h after transfection. Four firefly luciferase reporter vectors with a modified reporter gene and vector backbone. Stay notified of promega events, products and news. All sirnas, the mir520b inhibitor, and the control inhibitor were purchased from. 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.
Luciferase activity was detected 48 h after transfection. 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. Dna sequencing confirmed the identity of all constructs. The pgl3basic vector without an insert sequence was used as a negative control. Duelluciferase reporter assay the wide type and the mutation of the promoter segment sequences of integrin.
427 464 692 1495 1417 1342 999 1050 77 1443 90 730 540 412 717 16 1131 237 440 1470 742 638 899 49 1371 566 437 1489 630 1214 84 1236 121 556 217 1423 839 405 112 290 318