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Finally, the possible patho-physiological significance of the +190G/C polymorphism was further confirmed by detailed functional assays in both endothelial and non-endothelial cell lines with consistent results.
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Luciferase reporter assays showed that the +190C allele resulted in 14% ∼45%reduction in luciferase expression in endothelial and non-endothelial cells when compared with the +190G allele.
In a transfection assay, the C allele of rs2236659 showed a 37 40% increase in luciferase expression of the reporter gene luciferase in endothelial and non-endothelial cells compared with the T allele.
This is most likely due to differences between endothelial and non-endothelial cells [ 20].
The first stage identified endothelial genes by comparing the expression patterns of genes between endothelial and non-endothelial cell lines.
Using a custom relational database developed in house, cDNA libraries were collected and divided into 2 pools (endothelial and non-endothelial cells respectively) and formatted into BLAST databases.
These genes were then micro-arrayed and probed with RNA samples from a selection of cultured endothelial and non-endothelial cell types.
TEM1 (endosialin or CD 248) [ 14] has a count of 1 and 2 ESTs for the endothelial and non-endothelial pools respectively.
The HSPA1A 5'-flanking region bearing the −110C/+190C haplotype exhibited about 30% ∼50%lower RLA than the −110A /+190G haplotype in endothelial (HUVEC) and non-endothelial cells (Hela).
We determined whether any given ortholog regulated during CAM maturation had preferential expression in endothelial cells compared to non-endothelial cells using a recently published in silico method, which compares sequence of any given gene to two pools of EST cDNA libraries, endothelial ESTs and non-endothelial ESTs, followed by an FDR-based approach [32].
To verify that tamoxifen treatment of Rac1 flox/flox PDGFB-iCreER endothelial cells induced Rac1-deletion, endothelial cells and non-endothelial cells were isolated from Rac1 flox/flox PDGFB-iCreER mice and treated with tamoxifen (OHT) or ethanol (vehicle) in culture.
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