The CMV promoter fragment was PCR amplified from pEGFPC3 vector (Clontech) and cloned between the EcoRI and BamH I sites of the promoter-less pAcGFP1-1 vector (Clontech) to derive the pCMV-AcGFP1-1 plasmid. 3L-S and 3L-L regions from the DNMT3L locus (figure 1 ) were PCR amplified from the HeLa genomic DNA using the primers 3LF (5′-CCTGAGGGCCCCATCCTCTG-3′ ) and 3LSR (5′-AAGGATCCAGGCCCACCTGGGAC-3′ ) for 3L-S and 3LF and 3LLR (5′-CAGGGACCCCTGGGGATGGTCTTGGCC-3′ ) for 3L-L. The fragments were cloned in both orientations upstream of the CMV promoter in pCMV-AcGFP1-1 vector between XhoI and EcoRI. As a negative control we cloned a 1 kb region from human chromosome 1, which was previously shown to have no transcriptional potential upstream of the CMV promoter [18] (link). A 1.5 kb region from the H19 ICR, which has been shown to be a transcriptional repressor [19] (link) was also cloned upstream of the CMV promoter (Figure 1 ). As a control for transfection efficiency we used the pG5luc vector (Promega) containing the luciferase reporter gene. Transfections were done in HEK293 cells using Lipofectamine 2000 (Invitrogen) in duplicates following the manufacturer's protocol.
Pg5luc vector
Manufactured by Promega
The PG5luc vector is a plasmid that contains the firefly luciferase gene under the control of a minimal promoter and enhancer elements. This vector is commonly used in reporter gene assays to study gene expression and regulation.
Automatically generated - may contain errors
Lab products found in correlation
Paav mincmv mcherry vector, by Addgene (2 mentions)
Pegfp c3 vector, by Takara Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by PromoCell (1 mentions)
Endothelial cell growth medium, by PromoCell (1 mentions)
Pbind vector, by Promega (1 mentions)
Pact vector, by Promega (1 mentions)
Luciferase reporter assay kit, by Vazyme (1 mentions)
6 protocols using pg5luc vector
1
Cloning DNMT3L Regulatory Regions
2
Cell Culture Protocols for Diverse Cell Lines
3
Yeast Two-Hybrid Assay for Protein Interactions
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pBIND vector (Promega) as a bait that includes the GAL4 DNA-binding domain and pACT vector (Promega) as a prey that includes the VP16 activation domain were used for plasmid construction. Open reading frames of TLP/mutant and p53/mutant were linked just downstream from the GAL4 DNA-binding domain of pBIND and VP16 activation domain of pACT vector, respectively. pG5-luc vector (Promega) was used as a reporter plasmid with the luciferase reporter gene.
4
Cloning TIMP Promoters for Reporter Analysis
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A 1900 bp TIMP-1 promoter sequence spanning from 1026 bp upstream of the start ATG through the 9 bp of exon 2 was synthesized (GenScript, Piscataway, NJ, USA) and subcloned into the pGL4.10 (Promega, Madison, WI, USA) promoter-less firefly luciferase reporter vector. A similar promoter sequence driving the expression of a lacZ reporter was shown to reproduce the spatial and temporal expression patterns of TIMP1 gene in a mouse embryo.15 (link) TATA-containing 1158 bp TIMP-2 and 1154 bp TIMP-3 promoter sequences located immediately upstream of the start codon reported to exhibit promoter activity16 (link),17 (link) were synthesized and subcloned upstream of firefly luciferase reporter vector. The promoter sequence for mouse TIMP-2 was deduced from the human sequence. The 3 TIMP promoter sequences are listed in Table S1 . The 5xGAL4 sequence polymerase chain reaction (PCR) amplified from the pG5luc vector (Promega) flanked by the Mlu I and Bam HI restriction enzyme sites was subcloned upstream of the minCMV.mCherry sequence in the pAAV-minCMV-mCherry vector (Addgene #27,970) to create the control GAL4-mCherry reporter.
5
Cloning TIMP Promoters for Reporter Analysis
6
Mammalian Two-Hybrid Assay for HMGB1-HDAC1 Interaction
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Mammalian two‐hybrid assays were performed as previously reported. Briefly, HMGB1 and HDAC1 cDNA were cloned into the pBIND and pACT vectors, then co‐transfected with the pG5luc vector (Promega). After 48 h, reporter activity was measured using the Luciferase Reporter Assay Kit (Vazyme), and Renilla activity was used to normalize firefly luciferase activity.
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