pcmv sport β-galactosidase control vector  (Thermo Fisher)

Journal: BMC Molecular Biology

Article Title: Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions

doi: 10.1186/1471-2199-8-36

Figure Lengend Snippet: Sp1 and Sp3 are antagonistic at the topoisomerase IIα promoter. All transfections were performed in triplicate with 1.0 μg of -617 wt topoisomerase IIα reporter plasmid and 1 μg of pCMV Sport β-galactosidase control vector. Relative luciferase activity was calculated by arbitrarily setting the control (0 μg, containing only topoisomerase IIα reporter plasmid and pCMV Sport β-galactosidase control vector) to 100%. Results are expressed as ± SEM where n = 9, *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: Transient transfection assays were carried out in 12-well tissue culture plates with cells at approximately 60% confluence using 1 μg of reporter vector (topoisomerase IIα-617pGL3Basic [ ], 1 μg of pCMV Sport β-galactosidase control vector (Invitrogen) and various amounts of Sp1, and/or Sp3, co-expression vectors using Metafectene (Biontex) transfection reagent according to the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: BMC Molecular Biology

Article Title: Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions

doi: 10.1186/1471-2199-8-36

Figure Lengend Snippet: Sp3 maintains transcriptional repression in the presence of Sp1. All transfections were carried out in triplicate with 1.0 μg of -617 wt topoisomerase IIα reporter plasmid and 1 μg of pCMV Sport β-galactosidase control vector. Relative luciferase activity was calculated by arbitrarily setting the control (no added Sp1 or Sp3) to 100%. Results are expressed as ± SEM where n = 9, *** p < 0.001, ** p < 0.01, and * p < 0.05

Article Snippet: Transient transfection assays were carried out in 12-well tissue culture plates with cells at approximately 60% confluence using 1 μg of reporter vector (topoisomerase IIα-617pGL3Basic [ ], 1 μg of pCMV Sport β-galactosidase control vector (Invitrogen) and various amounts of Sp1, and/or Sp3, co-expression vectors using Metafectene (Biontex) transfection reagent according to the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: BMC Molecular Biology

Article Title: Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions

doi: 10.1186/1471-2199-8-36

Figure Lengend Snippet: Sp3 transcriptional repression is dominant over activation by Sp1. All transfections were performed in triplicate with 1.0 μg of -617 wt topoisomerase IIα reporter plasmid and 1 μg of pCMV Sport β-galactosidase control vector. Relative luciferase activity was calculated by arbitrarily setting the control (no added Sp1 or Sp3) to 100%. Results are expressed as ± SEM where n = 9, *** p < 0.001, ** p < 0.01, and * p < 0.05

Article Snippet: Transient transfection assays were carried out in 12-well tissue culture plates with cells at approximately 60% confluence using 1 μg of reporter vector (topoisomerase IIα-617pGL3Basic [ ], 1 μg of pCMV Sport β-galactosidase control vector (Invitrogen) and various amounts of Sp1, and/or Sp3, co-expression vectors using Metafectene (Biontex) transfection reagent according to the manufacturer's instructions.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Regulation of Krüppel-Like Factor 4 (KLF4) expression through the transcription factor Yin-Yang 1 (YY1) in non-Hodgkin B-cell lymphoma

doi: 10.18632/oncotarget.26745

Figure Lengend Snippet: ( A ) Two potential binding sites for the transcription factor KLF4 obtained after bioinformatics analysis using two online servers, JASPAR and TRANSFACT, are displayed. The region from −2000 to +160 bp in the KLF4 gene was analyzed for Site Transcription Initiation (SIT). A weight matrix obtained from the JASPAR database for the transcription factor KLF4 is displayed. ( B ) Putative binding YY1 sites in the KLF4 promoter that are involved in regulating expression. Transfection assays were performed using the PC3 cell line to assess the effects of directed mutagenesis at each of the YY1 binding sequences, located at sites -950 bp and -105 bp in the promoter region of the KLF4 gene. The schematic shows each of the mutated sites, and the graph indicates normalized luciferase reporter gene expression levels obtained by measuring β-galactosidase via co-transfection with a reporter gene plasmid (top panel); fold changes are reported (bottom panel). The results are representative of three independent experiments (one-way ANOVA, * p < 0.005, ** p < 0.001). ( C ) ChIP was conducted for each potential YY1 binding site in the KLF4 promoter. The results show that YY1 binds the promoter region of KLF4.

Article Snippet: PC3 cells were co-transfected with Lipofectamine 2000 (Invitrogen), following the manufacturer's instructions, with the reporter plasmid containing the wild-type promoter sequence of the promoter KLF4 (pGL3-KLF4-pro-luc) or different constructs modifying the YY1 mutant sites in the KLF4 promoter and pCMV-Sport-β-galactosidase vector (ThermoFisher Scientific), which is commonly used for determining transfection efficiency.

Techniques: Binding Assay, Expressing, Transfection, Mutagenesis, Luciferase, Cotransfection, Plasmid Preparation

Journal: Genetics

Article Title: Gene Regulatory Variation in Drosophila melanogaster Renal Tissue

doi: 10.1534/genetics.118.301073

Figure Lengend Snippet: Genetic differentiation in upstream regions and the effect of upstream variation on expression divergence. (A) Schematic of the reporter gene constructs. The upstream regions of six cytochrome P450 genes from the S58 and Z418 strains were cloned in front of the lacZ gene and integrated into the D. melanogaster genome. Lines indicate the polymorphic sites between S58 and Z418, with different colors indicating which strain has the ancestral variant (red = Z418, blue = S58, and gray = unknown). The total number of polymorphisms in each category is shown in the same color scheme to the right of the upstream region. (B) Relative expression of each endogenous gene in Malpighian tubules (MTs) of the S58 and Z418 parental strains (blue) and their alleles in the S58×Z418 hybrid (red) as determined by RNA-sequencing. Also shown is the relative expression of the respective reporter genes in MTs (orange), whole flies (white), and carcasses with the MTs removed (gray) as determined by β-galactosidase activity. Error bars represent SEM. For reporter genes, the significance of expression differences between the two upstream regions was assessed using a Student’s t-test. (C) Expression divergence in the MT between the Z418 and S58 strains partitioned according to type of regulatory variation. The contributions of cis variation within the tested upstream region (red), cis variation in other regions (gray), and trans variation (blue) were estimated from the RNA-sequencing and reporter gene data. For genes showing a nonadditive cis × trans interaction (Cyp12a5 and Cyp6a20), the total cis variance was assumed to be the maximum possible, i.e., the observed expression divergence between the parental strains. (D) The proportion of highly differentiated SNPs within the tested upstream regions relative to that of their respective chromosome arms. Values greater than one indicate an enrichment of SNPs with FST falling within the top 10% (red) or top 5% (blue) of the chromosomal FST distribution. Significance was assessed using a χ2-test. * P < 0.05, ** P < 0.01, and *** P < 0.005.

Article Snippet: A 3.5-kb Not I fragment of the pCMV-SPORT -β -gal vector (Invitrogen) containing the Escherichia coli lacZ gene was inserted into the NotI site downstream of the cloned upstream sequences in the TOPO vector.

Techniques: Expressing, Construct, Clone Assay, Variant Assay, RNA Sequencing Assay, Activity Assay