Mechanisms underlying the enhancing effect of the first intron on vWF^LacZ/+ gene expression. (A) Organs from vWF^LacZ/+ and vWFΔInt^LacZ/+ mice were assayed for LacZ mRNA expression by qPCR. The results show the mean ± standard deviation of mRNA expression (relative to vWF^LacZ/+ in brain) obtained in triplicate from 3 independent experiments. Br, brain; He, heart; Lu, lung; Li, liver; Ki, kidney; Di, diaphragm; SM, skeletal muscle; Ao, aorta; Sp, Spleen. (B) Bone marrow from vWF^LacZ/+ and vWFΔInt^LacZ/+ mice was assayed for LacZ mRNA expression by real-time PCR. The results show the mean ± standard deviation of mRNA expression (relative to vWF^LacZ/+) obtained in triplicate from 3 independent experiments. n.s., nonsignificant. (C) Endothelial cells were harvested from different organs from vWF^LacZ/+ and vWFΔInt^LacZ/+ mice using CD31-coated magnetic beads. RNA was isolated within 90 minutes and assayed for LacZ mRNA expression by qPCR. The results show the mean ± standard deviation of mRNA expression obtained in triplicate from 3 independent experiments. (D) ChIP assay was performed using double-sorted endothelial cells from the hearts of vWF^LacZ/+ and vWFΔInt^LacZ/+ mice. DNA was sheared and the resulting DNA-protein complexes were immunoprecipitated in the absence or presence of antibodies to PolII or control IgG. Real-time PCR analysis was performed using the precipitated DNA fragments and primers for the proximal promoter region. (E) mRNA in cytoplasmic and nuclear fractions or total mRNA was extracted from heart ECs of vWF^LacZ/+ and vWFΔInt^LacZ/+ mice. LacZ mRNA levels were measured by qPCR using gene-specific primers. The results show the mean ± standard deviation of mRNA expression (relative to vWF^LacZ/+) obtained from 5 independent experiments.