reporter plasmid cpg  (InvivoGen)

Journal: Disease Models & Mechanisms

Article Title: Physiological tissue-specific and age-related reduction of mouse TDP-43 levels is regulated by epigenetic modifications

doi: 10.1242/dmm.049032

Figure Lengend Snippet: In vitro methylation of the mouse Tardbp promoter. EEF1A1 and the empty pCpGfree-basic-Lucia vector were used as positive and negative controls, respectively. On the left of each quantification, cartoons of the constructs used in the assay are shown. White rectangles represent unmethylated CpG islands, and grey rectangles represent methylated CpG islands. Data are mean±s.e.m. ( n =3). Solid black circles represent individual data points. Pairwise comparison was performed using an unpaired two-tailed Student's t -test. *** P <0.001; **** P <0.0001; ns, not significant.

Article Snippet: The amplicon was subsequently inserted into the CpG-free and promoterless vector pCpGfree-basic-Lucia (InvivoGen, San Diego, CA, USA) upstream of the luciferase reporter gene upon introduction of a KpnI restriction site using the quick-change PCR mutagenesis technique.

Techniques: In Vitro, Methylation, Plasmid Preparation, Construct, Comparison, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Physiological tissue-specific and age-related reduction of mouse TDP-43 levels is regulated by epigenetic modifications

doi: 10.1242/dmm.049032

Figure Lengend Snippet: TARDBP methylation inversely correlates with human TDP-43 expression. (A) Cartoon of human TARDBP promoter predicted to contain 125 CpG sites, encompassed in three CpG islands. Numbering is in respect to the TSS. Bisulphite sequencing analysis of the promoter in SH-SY5Y cells showed the promoter to be strongly methylated in island one. Each circle represents a CpG site in island one or three. Solid dark circles represent sites where DNA was observed to be methylated. Each line represents a clone (six clones were sequenced). (B) Upper panel: representative western blots of TDP-43 and GAPDH in SH-SY5Y cells not treated (NT) and treated with 10 μM of 5-AZA for 48 or 72 h. Bar charts show the quantification after 48 h (dark grey bars) and 72 h (light grey bars) of protein (left) and mRNA (right). Values are normalized for GAPDH and expressed as fold over TDP-43 level in NT cells at 48 h ( n =3; for qPCR data, two technical replicates for each of three biological replicates were performed). Black circles represent individual data points. (C) In vitro methylation assay of the human TARDBP promoter. EEF1A1 and the empty pCpGfree-basic-Lucia vector were used as positive and negative controls, respectively. On the left of each quantification, cartoons of the constructs used in the assay are shown. White rectangles represent unmethylated CpG islands, and grey rectangles represent methylated CpG islands. Data are mean±s.e.m. ( n =3). Black circles represent individual data points. Pairwise comparison was performed using an unpaired two-tailed Student's t -test. * P <0.05; *** P <0.001; **** P <0.0001 ns, not significant.

Article Snippet: The amplicon was subsequently inserted into the CpG-free and promoterless vector pCpGfree-basic-Lucia (InvivoGen, San Diego, CA, USA) upstream of the luciferase reporter gene upon introduction of a KpnI restriction site using the quick-change PCR mutagenesis technique.

Techniques: Methylation, Expressing, Bisulfite Sequencing, Clone Assay, Western Blot, In Vitro, Plasmid Preparation, Construct, Comparison, Two Tailed Test