cpg free pcpgl basic vector  (New England Biolabs)

Journal: Acta Neuropathologica Communications

Article Title: Promoter DNA methylation regulates progranulin expression and is altered in FTLD

doi: 10.1186/2051-5960-1-16

Figure Lengend Snippet: GRN expression in human lymphoblast cell lines is inversely correlated to its promoter methylation. ( a ) GRN net secretion was measured by ELISA in LCLs derived from neurologically healthy individuals (LCL #1-13), unaffected relatives of FTLD patients (LCL #14, 15, highlighted in blue) and FTLD-patients (LCL#16, 17, highlighted in red). n = 3, mean ± SEM. ( b ) Scheme of GRN promoter region. Red bars depict PCR-amplicons analyzed for DNA methylation levels by MassARRAY. Blue bars indicate full length and short promoter region that was cloned into the pCpGL vector for luciferase assays (compare Figure ). White circles display CpG units in amplicons A-1 to A-5 and A-DAC quantified by MassARRAY. CpG units that were not analyzed are not shown. Asterisks indicate significant correlation between GRN mRNA expression or GRN secretion and GRN methylation at respective CpG unit (*p < 0.05, linear regression analysis, Benjamini Hochberg multiple testing and FDR correction, compare Additional file : Table S3). ( c ) Average DNA methylation levels in amplicons A-1 to A-5 for individual LCLs are plotted. Mean ± SD. Color code as in (a). ( d ) Correlation between GRN mRNA expression and average DNA methylation at CpG units 1, 2, 6, 8 and 11 is shown. GRN mRNA expression was quantified by qPCR and normalized to PGK1 and GAPDH. Relative mRNA expression levels were plotted against average DNA methylation levels. Correlation between parameters was quantified by linear regression analysis, r 2 and p-values are given. Color code as in (a). ( e ) Correlation between GRN secretion and average DNA methylation at CpG units 1, 2, 6, 8 and 11. GRN secretion was determined by ELISA and relative units (R.U.) were plotted against average DNA methylation levels. Correlation between parameters was quantified by linear regression analysis, r 2 and p-values are given. Color code as in (a).

Article Snippet: The GRN promoter region (−2423 to +207 bp relative to transcriptional start site, for primers see Additional file : Table S1) was cloned directly into the CpG-free vector pCpGL and in vitro methylated using recombinant DNA methyltransferase M.SssI (New England Biolabs).

Techniques: Expressing, Methylation, Enzyme-linked Immunosorbent Assay, Derivative Assay, DNA Methylation Assay, Clone Assay, Plasmid Preparation, Luciferase

Journal: Acta Neuropathologica Communications

Article Title: Promoter DNA methylation regulates progranulin expression and is altered in FTLD

doi: 10.1186/2051-5960-1-16

Figure Lengend Snippet: DNA methylation inhibits GRN promoter activity at distinct CpG units. In vitro methylated and unmethylated pCpGL plasmids containing the GRN core promoter region driving expression of firefly luciferase were transiently cotransfected into ( a ) HEK 293FT cells and ( b ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. The full length GRN promoter pCpGL plasmid, a GRN promoter construct with site specific mutations of the significant CpG units in amplicons A-1 and A-2, and a short GRN promoter construct lacking amplicons A-1 and A-2 were transiently cotransfected into ( c ) HEK 293FT cells and ( d ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. Luciferase reporter activity was measured 48 h (a + c, HEK 293FT) or 72 h (b + d, neurons) after transfection. Relative luciferase activity was determined by normalizing firefly luciferase against Renilla luciferase activity. The empty vector was used as negative control. Mean ± SEM, n ≥ 3. ***p < 0.001, Student’s t-test, sign . significant.

Article Snippet: The GRN promoter region (−2423 to +207 bp relative to transcriptional start site, for primers see Additional file : Table S1) was cloned directly into the CpG-free vector pCpGL and in vitro methylated using recombinant DNA methyltransferase M.SssI (New England Biolabs).

Techniques: DNA Methylation Assay, Activity Assay, In Vitro, Methylation, Expressing, Luciferase, Plasmid Preparation, Construct, Transfection, Negative Control

Journal: Acta Neuropathologica Communications

Article Title: Promoter DNA methylation regulates progranulin expression and is altered in FTLD

doi: 10.1186/2051-5960-1-16

Figure Lengend Snippet: Overexpression of DNMT3a alters GRN promoter activity in primary cortical neurons and reduces GRN mRNA expression in LCLs. ( a ) pCpGL plasmid containing the GRN core promoter and a DNMT3a overexpression construct were transiently transfected in HEK 293FT cells (left panel) and in rat primary cortical neurons (right panel). Relative luciferase activity was determined by normalizing firefly luciferase against Renilla luciferase activity. Empty vectors were used as negative control. Firefly luciferase expression was significantly reduced upon DNMT3a overexpression. Mean ± SEM, n ≥ 3. ***p < 0.001, ANOVA with Tukey’s Multiple Comparison test. ( b ) Lentiviral expression of DNMT3a in LCLs #3 and #14. Overexpression was verified by qPCR five days after viral transduction. n = 5, mean ± SEM, *p < 0.05, Student’s t-test. ( c ) GRN mRNA expression levels were significantly reduced in DNMT3a overexpressing LCLs as quantified by qPCR and normalized to PGK1 expression levels. n = 5, mean ± SEM, *p < 0.05, Student’s t-test.

Article Snippet: The GRN promoter region (−2423 to +207 bp relative to transcriptional start site, for primers see Additional file : Table S1) was cloned directly into the CpG-free vector pCpGL and in vitro methylated using recombinant DNA methyltransferase M.SssI (New England Biolabs).

Techniques: Over Expression, Activity Assay, Expressing, Plasmid Preparation, Construct, Transfection, Luciferase, Negative Control, Comparison, Transduction