pgl3‐basic vector  (GeneBay Inc)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/JAHA.123.030460

Figure Lengend Snippet: A , Alignment of the human, mouse, and rat Ht sequences is shown. Conserved nucleotides are marked with asterisk. The arrow indicates the TSS. B through E , Luciferase assay in SH‐SY5Y cells transfected for 48 hour with pGL3‐Br ( B and D ) or pGL3‐Ht ( C and E ) and cotransfected with siCTL, siDNMT1, siMeCP2, or siREST ( B , C ), or with EV, DNMT1, MeCP2, or REST ( D and E ). * P ≤0.05 vs siCTL or EV by 1‐way ANOVA analysis followed by Tukey's post hoc test (n=4). CTL indicates control; DNMT1, DNA‐methyltransferase‐1; EV, empty vector; Ht , heart promoter, MeCP2, methyl‐CpG binding protein 2; NCX1, sodium/calcium exchanger 1; pGL3‐Br, Ncx1 brain promoter construct inserted in pGL3basic; pGL3‐Ht, Ncx1 heart promoter construct inserted in pGL3basic; REST, repressor element 1‐silencing transcription factor; si, small interfering; and TSS, transcription start site.

Article Snippet: The PCR product was purified using StrataPrep DNA gel extraction kits (Agilent, Milan, Italy) and cloned into multiple cloning sites of pGL3‐basic luciferase expression vector (Promega, Milan, Italy).

Techniques: Luciferase, Transfection, Plasmid Preparation, Binding Assay, Construct

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/JAHA.123.030460

Figure Lengend Snippet: A , Top: JASPAR matrix representation (MA0138.1) of the consensus REST binding site on Ht gene ( Ht ‐RE1). Ht ‐RE1 sequence is represented in International Union of Pure and Applied Chemistry code. Bottom: Partial human genomic Ht sequence containing the predicted REST binding site (underlined). B , Luciferase assay in SH‐SY5Y cells under the following experimental conditions: (1) pGL3basic, (2) pGL3‐Ht+EV, (3) pGL3‐Ht+REST, (4) pGL3‐Ht‐RE1mut+EV, (5) pGL3‐Ht‐RE1mut+REST. * P ≤0.05 vs pGL3Ht+EV by 1‐way ANOVA analysis followed by Tukey's post hoc test (n=4). C , ChIP with anti‐REST antibody followed by qPCR of the promoter region containing the RE1 site on the Ht gene in SH‐SY5Y cells transiently transfected with (1) pGL3‐Ht+EV, (2) pGL3‐Ht+REST, (3) pGL3‐Ht+RE1mut+REST. * P ≤0.05 vs pGL3‐Ht+EV by 1‐way ANOVA analysis followed by Tukey's post hoc test (n=4). ChIP indicates chromatin immunoprecipitation; CTL, control; DNMT1, DNA‐methyltransferase‐1; EV, empty vector; Ht , heart promoter, MeCP2, methyl‐CpG binding protein 2; NCX1, sodium/calcium exchanger 1; pGL3‐Br, Ncx1 brain promoter construct inserted in pGL3basic; pGL3‐Ht, Ncx1 heart promoter construct inserted in pGL3basic; qRT‐PCR, quantitative real time polymerase chain reaction; RE1, repressor element 1; and REST, repressor element 1‐silencing transcription factor.

Article Snippet: The PCR product was purified using StrataPrep DNA gel extraction kits (Agilent, Milan, Italy) and cloned into multiple cloning sites of pGL3‐basic luciferase expression vector (Promega, Milan, Italy).

Techniques: Binding Assay, Sequencing, Luciferase, Transfection, Chromatin Immunoprecipitation, Plasmid Preparation, Construct, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: A Functional Variant of the Dimethylarginine Dimethylaminohydrolase-2 Gene Is Associated with Insulin Sensitivity

doi: 10.1371/journal.pone.0036224

Figure Lengend Snippet: A. Quantification of DDAH2 mRNA levels in primary HUVECs naturally carrying the G/G (n = 10) or C/G genotype (n = 5). Total RNA was extracted from confluent cells. cDNA was reverse transcripted and analyzed by quantitative Real-Time PCR technique. Bars represent the means ± SD of two independent experiments carried out in triplicate (* P = 0.008 for G/G HUVECs vs C/G HUVECs); B. Luciferase activity was evaluated in immortalized HUVEC transfected with an empty PGL3 luciferase vector (PGL3, light gray bar, n = 21); or a PGL3 vector carrying the rs9267551 G (PGL3G, white bar, n = 21); or a PGL3 vector carrying the rs9267551 C form (PGL3C, black bar, n = 21). Luciferase activity in mock transfected cells (mock, dark gray bar, n = 21) was assessed as a control. (** P = 0.00076 for PGL3G vs. PGL3C).

Article Snippet: Both fragments were inserted in the pGL3 basic luciferase expression vector (Promega, Madison, WI) and constructs sequence was confirmed by direct sequencing.

Techniques: Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Plasmid Preparation