pgl2-sv40 vector  (Promega)

Journal: eLife

Article Title: Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction

doi: 10.7554/eLife.56697

Figure Lengend Snippet: ( A ) Basal activity of reference alleles of RE candidates in HL1 (light grey bars) and COS7 cells (dark grey bars). Luciferase activity for each RE candidate is normalized to the activity of the empty pGL2-SV40 vector. ( B ) Luciferase activity of reference alleles of RE candidates upon stimulation with Smad/Gata- (SG4) and Tcf+LiCl- (Wnt) factors in COS7 cells. Luciferase activity of each RE candidate is normalized to the activity of each RE candidate upon co-transfection with pcDNA (control). Transfections were performed in duplicates and replicated twice. Error bars represent standard deviation. *: p<0.05.

Article Snippet: Amplified sequences were ligated in the XcmI site of a modified pGL2-SV40 vector (Promega) using T4 DNA Ligase (ThermoFisher Scientific; 15224–090).

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Cotransfection, Transfection, Standard Deviation

Journal: eLife

Article Title: Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction

doi: 10.7554/eLife.56697

Figure Lengend Snippet: ( A ) Overview of the murine Tbx3 locus with ATAC_AVJ, EMERGE and STARR_SG4 data. Fragments selected for luciferase assay, selected either on STARR-seq activity or EMERGE/ATAC peaks are depicted below. ( B ) Correlation between luciferase reporter activity (fold change of reporter activity over activity of empty pGL2-SV40 vector; threshold FC >2) and STARR-seq (log2FC of SG4 over pcDNA control; threshold log2FC >0.58 (FC >1.5)) of selected fragments.

Article Snippet: Amplified sequences were ligated in the XcmI site of a modified pGL2-SV40 vector (Promega) using T4 DNA Ligase (ThermoFisher Scientific; 15224–090).

Techniques: Luciferase, Activity Assay, Plasmid Preparation

Journal: Biology of Reproduction

Article Title: Endocrine disruptor exposure during development increases incidence of uterine fibroids by altering DNA repair in myometrial stem cells †

doi: 10.1093/biolre/ioy097

Figure Lengend Snippet: DNA end-joining efficiency is decreased in DES vs. VEH MSCs. Luciferase gene reporter assay detected lesser DNA end-joining activity in DES vs. VEH MSCs following co-transfection with linearized HindIIIpGL2-control luc and CMV-β-gal plasmids. Means of combined experimental and biological replicates were compared using a two-tailed Student unpaired t-test. Lines represent the mean normalized end-joining activity ± SD. *P < 0.0001

Article Snippet: Plasmid DNA preparation Briefly, SV40- luc pGL2-control vector plasmid DNA ( Supplemental Figure S1A , Promega, Madison, WI) was used to transform DH5α Escherichia coli cells for plasmid amplification and was then isolated using PureYield Plasmid Midiprep System (Promega).

Techniques: Luciferase, Reporter Assay, Activity Assay, Cotransfection, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Myoferlin regulation by NFAT in muscle injury, regeneration and repair

doi: 10.1242/jcs.065375

Figure Lengend Snippet: The 1543-bp myoferlin promoter drives high-level expression. (A) Sequence conservation of the first 1500 bp of the myoferlin promoter from humans and mice. ‘0’ refers to the transcriptional start site and ‘275’ marks the initiator methionine. Regions as high as 75% similarity were identified. Below is a schematic of four different promoter constructs generated and tested. (B) Activity of the myoferlin promoter fragments shown in A in C2C12 myoblasts. The 1543-bp–pGL2 reporter activates luciferase expression 82-fold above background.

Article Snippet: The pGL2-basic vector or the pGL2-SV40 control vector (Promega, E1631) replaced the reporter vector as a negative or positive control, respectively.

Techniques: Expressing, Sequencing, Construct, Generated, Activity Assay, Luciferase

Journal: Journal of Cell Science

Article Title: Myoferlin regulation by NFAT in muscle injury, regeneration and repair

doi: 10.1242/jcs.065375

Figure Lengend Snippet: NFAT binds the myoferlin promoter and regulates its activity. (A) The position of the five predicted NFAT-binding sites are designated by arrows. The four different regions amplified in the ChIP assay are indicated below the line. (B) ChIP assays using anti-NFAT antibodies for endogenous NFAT proteins from confluent C2C12 cells. NFATc1, NFATc2 and NFATc3 were tested, and NFATc3 was detected occupying all NFAT sites tested. Low-level occupancy of NFATc1 was also detected. NFATc2 was not detected at the myoferlin promoter. (C) pGL2 constructs were generated containing mutations in five predicted NFAT-binding sites in the 1543-bp myoferlin promoter labeled NFAT site 1 through 5. Constructs were transfected into C2C12 myoblasts and luciferase expression analyzed. Mutagenesis of NFAT sites 1 and 3 reduced luciferase expression by approximately 30% and 50%, respectively, compared with the 1543-bp–pGL2 construct or mutants 2, 4 or 5 (*P<0.004). (D) The 1543-bp–pGL2 reporter construct or pGL2-basic control vectors were transfected into C2C12 myoblasts in combination with activated calcineurin A (CnA), NFATc1 and/or NFATc3 expression plasmids. Luciferase expression was increased by twofold with the addition of CnA, NFATc1 and NFATc3 compared with the 1543-bp–pGL2 vector alone (P<0.0001). (E) The 1543-bp–pGL2 construct was transfected into C2C12 myoblasts and, 18 hours post-transfection, 1 μM or 5 μM CsA was added to the cultures. After 24 hours, luciferase expression was analyzed. The addition of 5 μM CsA reduced the 1543-bp myoferlin-promoter activity by 30% compared with 1543-bp–pGL2 (*P<0.05).

Article Snippet: The pGL2-basic vector or the pGL2-SV40 control vector (Promega, E1631) replaced the reporter vector as a negative or positive control, respectively.

Techniques: Activity Assay, Binding Assay, Amplification, Construct, Generated, Labeling, Transfection, Luciferase, Expressing, Mutagenesis, Plasmid Preparation