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Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A

y to some promoter elements but not others. We used the ChIP assay (Figure 1) to directly assess the binding of Pdx1 to promoter elements in vivo in Pdx1-transfected NIH 3T3 cells (NIH 3T3/ Pdx1). As a negative control for the immuno-precipitations, we performed identical experiments in NIH 3T3 cells transfected with an empty vector (NIH 3T3/EV). By coupling the ChIP assay to real-time PCR using SYBR Green I and the iCycler iQ detection system, we were able to reproducibly quantitate relative occupancy by Pdx1 to several β-cell specific and nonspecific promoters.

Immunoprecipitated samples were subject to quantitative real-time PCR using primers to amplify the β-cell-specific mouse I insulin, Glut2, IAPP, glucokinase, Pdx1, and Pax4 gene promoters, and the non-β-cell-specific glucagon and albumin gene promoters. Primer pairs (see table) were designed to amplify specific promoter regions that are believed either to be bound and regulated by Pdx1 (promoters for mouse I insulin, Glut2, IAPP, Pdx1, Pax4, and glucokinase genes) or to contain important regulatory regions with potential Pdx1 binding sites (promoter for glucagon gene). As a negative control for binding, the albumin promoter fragment was chosen as a random genomic element containing Pdx1 binding sites, but not believed to be regulated by Pdx1. The promoter fragments were amplified as homogeneous products of expected sizes, as shown in Figure 2.

As an example, Figure 3 shows the results (in duplicate determinations) of the amplification of the mouse I insulin promoter from NIH 3T3/Pdx1 and NIH 3T3/EV cells following ChIP. The data demonstrate that the NIH 3T3/Pdx1 chromatin that has been immunoprecipitated using Pdx1 ant
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