reporter plasmid cpg  (InvivoGen)

Journal: The Innovation

Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo

doi: 10.1016/j.xinn.2025.101151

Figure Lengend Snippet: Epigenetic silencing of the Gapdh-Snrpn-GFP reporter and endogenous CD151 in HEK293T cells using plasmid-based CRISPR OFF-EEs (A) Schematic of the epigenetic silencing strategy (SpCas9/SF01-OFF-EE). The Gapdh-Snrpn-GFP reporter is targeted by a catalytically inactive Cas protein (dSpCas9 or dSF01) fused to epigenetic editor (EE) domains (DNMT3A-3L and KRAB). The complex is directed by a specific single guide RNA (sgRNA or crRNA) to the Snrpn promoter, leading to targeted DNA methylation and silencing of GFP expression. (B) Representative flow cytometry plots showing GFP expression 30 days post-transfection with the indicated editor plasmids and a single corresponding gRNA. (C) Quantification of GFP silencing from experiments in (B). Data represent the mean ± SD of three independent biological replicates. (D) Methylation levels of individual CpG sites within the targeted Snrpn promoter and the non-targeted adjacent Gapdh promoter, confirming the specificity of methylation. Data are the mean ± SD ( n = 3). (E) Quantification of CD151 surface protein knockdown in HEK293T cells 30 days after transfection with editor plasmids and a pool of three guides. Data represent the mean ± SD ( n = 3). (F) Methylation levels of individual CpG sites within the targeted CD151 promoter. Data are the mean ± SD ( n = 3).

Article Snippet: The lentiviral plasmid for generating the Gapdh-Snrpn-GFP reporter cell line was also sourced from Addgene (Addgene, #70148).

Techniques: Plasmid Preparation, CRISPR, DNA Methylation Assay, Expressing, Flow Cytometry, Transfection, Methylation, Knockdown

Journal: Oncotarget

Article Title: Phenotypic alteration of CD8 + T cells in chronic lymphocytic leukemia is associated with epigenetic reprogramming

doi: 10.18632/oncotarget.9941

Figure Lengend Snippet: ( A ) Luciferase reporter assays: a 677 bp PDCD1 distal upstream DNA fragment was cloned into luciferase reporter vector pGL4.23 with a minimal promoter. Additionally, a version of the reporter plasmid was methylated by the M.SssI enzyme in vitro . The methylated, unmethylated, and empty reporter plasmids were then transfected into Jurkat cells to determine enhancer-mediated transcription. Schematic diagrams of experiment designs were shown on the left. (Errors bars = S.E.M.; * p < 0.05; ** p < 0.01). ( B ) Surface expression of PD-1 was upregulated in normal CD8 + T cells upon stimulation by PMA/ionomycin. The mean fluorescence intensity (MFI) was measured by flow cytometry. ( C and E ) DNA methylation level of the PDCD1 distal upstream region was measured by bisulfite pyrosequencing in purified naïve, memory CD8 + T cells and activated naïve CD8 + T cells from two independent ND samples. The first 7 CpG sites are the same CpGs analyzed in the far right panel of Figure . ( D and F ) qRT-PCR analysis of PDCD1 mRNA expression in purified naïve, memory CD8 + T cells and activated naïve CD8 + T cells from two ND samples. The relative expression of PDCD1 is calculated as fold changes using the ΔΔCt method. Naïve CD8 + T cells were activated by anti-CD3/anti-CD28 antibodies in the presence of IL2 for 6 days and then harvested for methylation and gene expression analysis.

Article Snippet: To test the effect of CpG methylation on the reporter activity, we methylated the reporter plasmid DNA in vitro using CpG Methyltransferase M.Sss I (NEB).

Techniques: Luciferase, Clone Assay, Plasmid Preparation, Methylation, In Vitro, Transfection, Expressing, Fluorescence, Flow Cytometry, DNA Methylation Assay, Purification, Quantitative RT-PCR

Journal: British Journal of Clinical Pharmacology

Article Title: Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects

doi: 10.1111/bcp.12675

Figure Lengend Snippet: DNA methylation status of three CpG islands in the ABCB1 promoter region in the stool DNA of healthy male Chinese volunteers. (A) Computational analysis of the CpG island distribution in the ABCB1 promoter region; (B–E) Distribution of the methylation levels of the ABCB1 promoter in individuals in total studied region, CpG island 1, CpG island 2 and CpG island 3, respectively. TSS, transcriptional start site.

Article Snippet: Whole ABCB1 reporter plasmids were methylated using CpG methyltransferase ( M . Sss I) (New England Biolabs, Massachusetts, USA), and in a parallel control reaction the same plasmid was mock-methylated in the absence of S -adenosylmethionine.

Techniques: DNA Methylation Assay, Methylation

Journal: British Journal of Clinical Pharmacology

Article Title: Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects

doi: 10.1111/bcp.12675

Figure Lengend Snippet: Methylation effects of different ABCB1 promoter regions on transcriptional activity in a luciferase reporter assay. The mean reporter activity ± standard error (Firefly/Renilla luciferase activity) from three independent experiments is presented.

Article Snippet: Whole ABCB1 reporter plasmids were methylated using CpG methyltransferase ( M . Sss I) (New England Biolabs, Massachusetts, USA), and in a parallel control reaction the same plasmid was mock-methylated in the absence of S -adenosylmethionine.

Techniques: Methylation, Activity Assay, Luciferase, Reporter Assay

Journal: British Journal of Clinical Pharmacology

Article Title: Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects

doi: 10.1111/bcp.12675

Figure Lengend Snippet: Bisulfite sequencing results of the ABCB1 promoter in stool DNA from healthy male Chinese subjects in the low and high methylation groups. Each row of circles represents a single cloned allele and each circle represents a single CpG dinucleotide. White circles represent unmethylated CpG dinucleotide, and black circles represent methylated CpG dinucleotide.

Article Snippet: Whole ABCB1 reporter plasmids were methylated using CpG methyltransferase ( M . Sss I) (New England Biolabs, Massachusetts, USA), and in a parallel control reaction the same plasmid was mock-methylated in the absence of S -adenosylmethionine.

Techniques: Methylation Sequencing, Methylation, Clone Assay

Journal: British Journal of Clinical Pharmacology

Article Title: Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects

doi: 10.1111/bcp.12675

Figure Lengend Snippet: High ABCB1 promoter methylation is associated with H3 histone deacetylation in the colonic epithelial cells of healthy male Chinese subjects. (A) Relative acetylated H3 enrichment. (B) Relative acetylated H4 enrichment. The results are expressed as the percentage of immunoprecipitate over total input DNA utilized. Error bars show the standard error of three different experiments with independent chromatin preparations. N = 15 per group, *P < 0.05.

Article Snippet: Whole ABCB1 reporter plasmids were methylated using CpG methyltransferase ( M . Sss I) (New England Biolabs, Massachusetts, USA), and in a parallel control reaction the same plasmid was mock-methylated in the absence of S -adenosylmethionine.

Techniques: Methylation

Journal: British Journal of Clinical Pharmacology

Article Title: Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects

doi: 10.1111/bcp.12675

Figure Lengend Snippet: The relationship between epigenetic status of the ABCB1 promoter and ABCB1 mRNA expression in vivo. (A) Interindividual variability in ABCB1 mRNA expression in colonic epithelial cells. (B) The epigenetic status of the ABCB1 promoter is related to the ABCB1 mRNA expression level in colonic epithelial cells. Error bars show the standard error of three different experiments with independent chromatin preparations. N = 15 per group; *P < 0.05.

Article Snippet: Whole ABCB1 reporter plasmids were methylated using CpG methyltransferase ( M . Sss I) (New England Biolabs, Massachusetts, USA), and in a parallel control reaction the same plasmid was mock-methylated in the absence of S -adenosylmethionine.

Techniques: Expressing, In Vivo

Journal: British Journal of Clinical Pharmacology

Article Title: Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects

doi: 10.1111/bcp.12675

Figure Lengend Snippet: The relationship between epigenetic status of the ABCB1 promoter and digoxin pharmacokinetics. (A) The epigenetic status of the ABCB1 promoter is related to the concentration–time profile of digoxin in healthy Chinese subjects. (B) The epigenetic status of the ABCB1 promoter is related to peak concentration values for digoxin in healthy Chinese subjects. Data show mean ± standard error; N = 15 per group.

Article Snippet: Whole ABCB1 reporter plasmids were methylated using CpG methyltransferase ( M . Sss I) (New England Biolabs, Massachusetts, USA), and in a parallel control reaction the same plasmid was mock-methylated in the absence of S -adenosylmethionine.

Techniques: Concentration Assay

Journal: Nucleic Acids Research

Article Title: Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

doi: 10.1093/nar/gku109

Figure Lengend Snippet: Analysis of TALE-VP64s targeting human and mouse Oct4 promoters. (A) Schematic representation of TALE-VP64 fusion proteins and mOct4/hOCT4-Luciferase ( Luc ) reporters. TALE domain was fused with NLS, VP64 and HA tag under the control of tetO-CMV promoter (upper panel). Luciferase reporters contained UAS element and mOct4/hOCT4 promoter (lower panel). (B) Luciferase activity of mouse Oct4 promoter induced by various TALE-VP64s (mO1–mO26). Schematic diagram showed the relative target locations of mO1–mO26 within the 2.3-kb regulatory region upstream of the TSS in mouse Oct4 gene. Effects of individual mOs were examined via co-transfection with mOct4-Luc reporter plasmid into HEK293T cells and relative luciferase activity was measured at 48 h after transfection. (C) Dox-dependent activity of mO21 and mO22 TALE-VP64s. Individual mOs were co-transfected with mOct4-Luc reporter plasmid into HEK293T cells, in the presence or absence of Dox. Relative luciferase activity was measured at 48 h after transfection. (D) TALE-KRAB fusion proteins mO21 KRAB or mO22 KRAB were co-transfected with mOct4-Luc reporter plasmid into E14 mouse ESCs, and relative luciferase activity was measured at 48 h after transfection. (E) Effect of positions on the activity of TALE-VP64s. TALE-VP64s mO6, mO11, mO17, mO20 and mO25 were co-transfected with wild-type mOct4-Luc reporter, reporters with point-mutation on the original target sequences of corresponding TALE-VP64s (constructs M-6, M-11, M-17, M-20 and M-25), and reporters with these target sequences relocated to the −120- to −104-bp region (constructs MR-6, MR-11, MR-17, MR-20 and MR-25), to assess their ability for activating the luciferase reporter. Relative luciferase activity was measured at 48 h after co-transfection. (F) Luciferase activity of human OCT4 promoter induced by TALE-VP64s targeting its CR1 and non-conserved region. TALE-VP64 hO1–hO8 and their target sites were illustrated. Relative luciferase activity was measured at 48 h after co-transfection with hOCT4-Luc reporter plasmid into HEK293T cells. (G) Schematic diagram of hNANOG-Luc reporter and its relative luciferase activity induced by TALE-VP64s (N1–N4). Data were shown as mean± SEM ( n = 3). * p < 0.05.

Article Snippet: In vitro methylation of mOct4-Luc and hOCT4-Luc reporter plasmids was performed using CpG methyltransferase M.SssI (New England Biolabs) as previously described ( ).

Techniques: Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Mutagenesis, Construct

Journal: Nucleic Acids Research

Article Title: Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

doi: 10.1093/nar/gku109

Figure Lengend Snippet: Activation of silenced Oct4 genes in mouse and human somatic cells by TALE-VP64s. (A) TALE code and variants of mO21 targeting potential mC using NG at the fourth, eighth or both nucleotides in its target sequences. ‘T’ in bold indicates the use of TALE code NG (T/mC) in the place of original code HD (C). TALE-VP64 mO21 Δ containing TALE code NI (A) in the place of HD (C) at the fourth and eighth nucleotide positions was included as negative control. (B) Activity of TALE-VP64 mO21 and its variants for activating unmethylated (upper panel) and methylated (lower panel) mOct4-Luc reporter. Individual TALE-VP64s were co-transfected with in vitro methylated/unmethylated reporter plasmids into HEK293T cells. Relative luciferase activity was measured at 48 h after transfection. (C) Activation of endogenous mouse Oct4 gene by TALE-VP64 mO21 and its variants (upper panel). NIH3T3 cells were transfected and harvested at 48 h after transfection. Relative Oct4 mRNA level was examined using qRT-PCR. The methylation status of endogenous Oct4 promoter in NIH3T3 cells was analyzed using bisulfite sequencing (lower panel). Mouse ESCs E14 were included as a control. The grey bar in the schematic diagram indicates the analyzed region (from −463 to −33 bp). Grey squares represent unmethylated CpG di-nucleotides and black squares represent methylated ones. The positions of the fourth and eighth nucleotide of the mO21 target sequence are indicated by asterisks. (D) TALE-VP64 hO3* was designed to target potential mC using NG at the sixth position in its target sequence (upper panel). The DNA methylation status of human OCT4 promoter in HEK293T cells was analyzed using bisulfite sequencing (lower panel). Human ESCs H1 were included as control. Grey squares indicate unmethylated CpG dinucleotides and black squares indicate methylated CpG di-nucleotides. Cytosine in the sixth nucleotide of hO3 target sequence is indicated with a asterisk. The analyzed region (from −217 to −32 bp) is indicated as grey bar in the schematic diagram. (E) Effects of hO3 and hO3* on unmethylated (left panel) and methylated (right panel) hOCT4-Luc reporters. Individual TALE-VP64s were co-transfected with in vitro methylated/unmethylated hOCT4-Luc reporters into HEK293T cells. Relative luciferase activity for each sample was examined at 48 h after transfection. (F) Activation of endogenous human OCT4 gene by hO3 and hO3*. qRT-PCR analysis was performed at 48 h after transfection in HEK293T cells. (G) Analysis of combinations of TALE-VP64 mOs for activating the endogenous mouse Oct4 gene in NIH3T3 cells. Combinations of mOs targeting CR4 (mO5, 6, 7, 8), non-conserved regions upstream of CR4 (up CR4) (mO1, 2, 3, 4), CR2 (mO9, 10, 11, 12, 13, 14, 15, 16), non-conserved region between CR2 and CR1 (NC) (mO17, 18, 19, 20, 21, 22) as well as CR1 (mO23, 24, 25, 26) were analyzed separately as well as in combination. Pool-1: all mOs targeting CR1, CR2 NC and CR4; Pool-2: mO10, 11, 16, 17, 19, 20, 21*, 22 and 25. qRT-PCR were performed at 48 h after transfection. (H) Analysis of various combinations of TALE-VP64 hOs for activating the endogenous human OCT4 gene in HEK293T cells. Significant transcriptional synergy was observed with multiple combinations. (I) OCT4 proteins were detected in HEK293T cells at 48 h after transfection with single or combinations of TALE-VP64 hOs. Western blot was performed with anti-OCT4. GAPDH was included as control for equal loading. Data were shown as mean± SEM ( n = 3).* P < 0.05.

Article Snippet: In vitro methylation of mOct4-Luc and hOCT4-Luc reporter plasmids was performed using CpG methyltransferase M.SssI (New England Biolabs) as previously described ( ).

Techniques: Activation Assay, Negative Control, Activity Assay, Methylation, Transfection, In Vitro, Luciferase, Quantitative RT-PCR, Methylation Sequencing, Sequencing, DNA Methylation Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

doi: 10.1093/nar/gku109

Figure Lengend Snippet: Activation of mouse and human Oct4 promoters by sgRNA-guided dCas9-VP64s. (A) Schematic representation of dCas9-VP64 and dCas9-KRAB fusion proteins. (B) Schematic diagram of eight sgRNAs (T1–T4, NT1–NT4) and their target sites in mouse Oct4 promoter. Effects of individual sgRNAs were examined via co-transfection of full-length dCas9-VP64 and mOct4-Luc reporter into HEK293T cells. sgRNA targeting GFP (sgGFP) was included as a negative control. Relative luciferase activity was measured at 48 h after transfection. (C) Analysis of dCas9 protein. Full-length dCas9 protein, dCas9 protein with deletion of N-terminal RuvC1 domain (ΔN) or deletion of C-terminal HNH domain (ΔC) were generated (left panel) and fused to VP64 or KRAB separately. Individual fusion proteins were co-transfected with sgRNA T2 and mOct4-Luc reporter. VP64 fusion proteins were examined for their ability to mediate sgRNA T2-guided transcriptional activation of mOct4-Luc reporter in HEK293T cells (middle panel) and KRAB fusion proteins were examined for their ability to mediate sgRNA T2-guided transcriptional repression of mOct4-Luc reporter in mouse ESCs E14 (right panel). Relative luciferase activity was measured at 48 h after transfection. (D) Analysis of combinatory effects of sgRNAs. Single or combined sgRNAs were co-transfected with dCas9-VP64 and mOct4-Luc reporter into HEK293T cells. Relative luciferase activity was measured at 48 h after transfection. Combination of sgRNA (NT1–NT4), as well as sgRNA (T1, T2, NT2 and NT3) showed significant synergistic effect in activating transcription of mOct4-Luc reporter. (E) Activation of endogenous mouse Oct4 genes by sgRNA-guided dCas9-VP64s. Individual or combined sgRNAs were co-transfected with dCas9-VP64 into NIH3T3 cells. Up-regulation of endogenous Oct4 genes was examined by qRT-PCR analysis at 48 h after transfection. (F) Schematic diagram of seven sgRNAs and their corresponding target sites in human OCT4 promoter (upper panel). Effects of individual and combined sgRNA were examined by luciferase assay (lower panel). HEK293T cells were co-transfected with sgRNA(s), dCas9-VP64 and hOCT4-Luc reporter and relative luciferase activity was measured at 48 h after transfection. Combination of sgRNAs H3 and H4, as well as H1–H4 showed a significant synergistic effect in activating transcription of hOCT4-Luc reporter. (G) Activation of endogenous human OCT4 gene by sgRNA-guided dCas9-VP64s. Individual or combined sgRNAs were transfected with dCas9-VP64s into HEK293T cells. Up-regulation of endogenous OCT4 gene was examined using qRT-PCR analysis at 48 h after transfection. (H) OCT4 proteins were detected in HEK293T cells at 48 h after transfection with dCas9-VP64 and combinations of sgRNAs (H3 and H4, or H1–H4). Western blot was performed with anti-OCT4. GAPDH was included as control for equal loading. (I) Combinatory effect of sgRNA/dCas9-VP64s and hO TALE-VP64s. sgRNAs H3 and H4 and TALE-VP64 (hO1, 3*, 4 and 6) were transfected together with dCas9-VP64 into HEK293T cells. Expression of endogenous OCT4 gene was examined using qRT-PCR at 48 h after transfection. Data were shown as mean± SEM ( n = 3).

Article Snippet: In vitro methylation of mOct4-Luc and hOCT4-Luc reporter plasmids was performed using CpG methyltransferase M.SssI (New England Biolabs) as previously described ( ).

Techniques: Activation Assay, Cotransfection, Negative Control, Luciferase, Activity Assay, Transfection, Generated, Quantitative RT-PCR, Western Blot, Expressing