Isolation and cloning of human vegf promoter region in. The suggestions are made assuming that you are using small promoter element in pgl3 basic vector. Xba i or spe i is compatible with the nhe i site of the pgl3 basic vector. 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 vector quantity is represented by a straight line segment, say. The cckar fragment was released from pcriitopocckar by speixhoi digestion followed by klenow blunting and subcloned into the nheixhoi klenowblunted site of pgl3 lucwpre to produce pgl3 cckarlucwpre cpluc and into the mscinhei klenow. The pgl3 basic vector contains no promoter or enhancer elements, so all of the basic transcroption sequences would need to be present to get luciferase. Such vectors belong to the foundation vector space rn of all vector spaces. 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. Introduction to vectors mctyintrovector20091 a vector is a quantity that has both a magnitude or size and a direction. A dioxin response element in the multiple cloning site of the. Both of these properties must be given in order to specify a vector completely. Reverse orientation of the promoter in the expression vector was obtained by creating a hindiii site at the 59end and xhoi site at the 39end by pcr using primer s and primer t table ii.
Functional analysis of promoter cpgmethylation using a cpgfree. Apr 26, 2016 the pgl3 control vector with the sv40 promoter upstream of the luc gene pgl3 sv40 and the promoterless pgl3 basic vector with the luc gene approx. We offer luciferase reporter reagents, multiple cloning site vectors, and flash, glow and dualspectral luciferase assay kits based on the intracellularly expressed firefly luciferase. 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. Use with snapgene software or the free viewer to visualize additional data and align other sequences. Isolation and cloning of human nqo1 promoter in pgl3 basic vector asma chinigarzadeh, razauden zulkifli, iman yaze and reyhaneh rahnamai tajadod. Basic vectors with no promoter that contain a multiple cloning region for cloning a promoter of choice. The transformation with pgl3 basic control vector works pretty well in all e. For example much of modern physics relies on vectors and the theory that describes them.
These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. 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 fragment of the ctsd promoter including two eboxes was cloned into pgl3 basic vector and transfected into tet21n mycn on, tet21n mycn off, mycnamplified imr575, mycn singlecopy shep and sy5y cells. 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. Basic operations with vectors this guide gives pictorial and algebraic explanations of how to add and subtract vectors and also how to multiply a vector by a scalar. 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. Functional analysis of promoter cpg methylation using a cpg. The saci fragment was subcloned into the pgl3 basic vector digested with saci. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. A dioxin response element in the multiple cloning site of the pgl3. All luciferase reporter plasmids were constructed using a pgl3basic vector. Endotoxin free tagless recombinant fgf21 was provided by antibody and immunoassay services ais, the university of hong kong. The pgl3basic vector lacks eukaryotic promoter and enhancer sequences. Promoter in reverse orientation pgl3 reverse 2652124.
At the end of your monthly term, you will be automatically renewed at the promotional monthly subscription rate until the end of the promo period, unless you elect to. For use with application quantitative analysis of factors that potentially regulate mammalian gene expression. The erk12 inhibitor pd98059 was obtained from sigma and dissolved in dimethyl sulfoxide dmso. The flavonoid quercetin induces ap1 activation in frtl5. This listings of restriction sites for the pgl2 luciferase reporter vectors are provided in sections 7. Circular intermediates of recombinant adenoassociated virus. Offer starts on jan 8, 2020 and expires on sept 30, 2020. Isolation and cloning of human vegf promoter region in pgl3. This article cites 32 articles, 16 of which you can access for free at. We use vectors to represent entities which are described by magnitude and direction. Pdf functional analysis of promoter cpgmethylation.
Description continued the pgl4 vector family includes. Lovely happy valentines day background with hearts. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The pgl4 luciferase reporter vectors are the next generation of reporter gene vectors optimized for more efficient expression in mammalian cells.
Problem with luciferase reporter assay researchgate. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. A vector is represented by a roman letter in bold face and its magnitude, by the same letter in italics. Introduction vectors are a very important idea in all areas of science.
Promega pgl3 luciferase reporter vectors pgl3 basic vector. A plant 35s camv promoter induces longterm expression of. 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. Hoechst 33342 alters luciferase gene expression in. Cell culture and adipocyte differentiationthe 3t3l1. Functional analysis of promoter cpgmethylation using a cpg free luciferase reporter vector. Promega pgl3 luciferase reporter vectors four firefly luciferase reporter vectors. Usa toll free in usa 8003569526 6082744330 fax 6082772516. Fibroblastgrowthfactor21inducesglucosetransporter1. Vector algebra 425 now observe that if we restrict the line l to the line segment ab, then a magnitude is prescribed on the line l with one of the two directions, so that we obtain a directed line segment fig 10. Hindiii a hindiii site is in the pgl3basic vector, and an. During ligation, the promoter was inserted in the reverse orientation of the pgl3 basic vector. For determining the role of the mcsdre in reporter activity, the mcsdre was deleted andor mutated in each plasmid by sitedirected mutagenesis.
This cd44 fragment includes the promoter region, exon1 and a part of intron1 of cd44. Numerous configurations of pgl4 vectors are available. The pgl3 basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Pdf functional analysis of promoter cpgmethylation using a. Home resources plasmid files luciferase vectors pgl4. In order to compute the coordinates ai the dual reciprocal basis ek is introduced in such a way that ek e i. When these constructs were transfected into murine nih 3t3 or human hek 293 cells for 24 h, the. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression, with a modified coding region for. The pgl3 bcl2 promotera construct was made by digesting the pgl3 bcl2 promoterfull plasmid with saci. Additionally, by uploading or otherwise transmitting material to any area of the web site you are granting the company a.
Supplemental data targeted expression of bikdd eradicates. In this unit we describe how to write down vectors, how to. Blackwell publishing asiafunctional roles of fli1, a member. Firefly luciferase assays and vectors thermo fisher. Basic vector alone and the pgl3control plasmid, which was used to. Molecular cloning and characterization of murine caspase. The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors. 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.
Functional analysis of promoter cpg methylation using a. 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. Insert transferred from pgem9zf vector to pgl3 basic vector. Values are the mean sd obtained from three independent experiments. Basic concepts a vector v in the plane or in space is an arrow. 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 pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. 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. High firefly luc activity in empty pgl3 basic vector reply. Thus, 9 2 3 may be viewed as a 1 3 matrix or a threedimensional row vector. Name vector type resistance marker bacterial resistance source sequence available.
Two arrows represent the same vector if they have the same length and are parallel see. This is a free resource for the scientific community that is compiled by addgene. A dualluciferase reporter assay was used to normalize for. It contains a zeocin resistance gene and the r6k origin of replication. In order to determine which vectors are most suitable for these types of analyses, vectors from promegas pgl3 and newly engineered pgl4 dualluciferase reporter assay. Ligation of vegf into pgl3 basic the digested pgl3 basic vector was treated with calf. The dimension of a row vector is the number of columns in the vector. As suggested before by others, pgl3 promoter vector has several cryptic sites and gives high. The specific transcriptional characteristics of the pgl3 vectors will vary for different cell types. The vector, including its multiple cloning site mcs, is completely free of cpg dinucleotides.
The pgl3 luciferase reporter vectors and the renilla luciferase vectors were. Thus, a directed line segment has magnitude as well as. Effect of vector backbone cpgmethylation on a cpgfree promoter in transient. Cathepsin d protects human neuroblastoma cells from. 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. This vector is engineered with fewer consensus regulatory sequences than the pgl3 vectors and a synthetic reporter gene, which has been codon optimized for mammalian expression. In analogous fashion, we can think of any vector with only one row a 1 n matrix as a row vector. Im trying clone a long insert into vector pgl3 researchgate. Pdf comparison of the responsiveness of the pgl3 and. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. Firefly luciferase is a 60kda protein produced in nature by several species of the lampyridae family of beetles which includes the genera photinus and luciola.
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