Deletion analysis using BAC-IRF-8 reporter constructs points to the role of the 3<sup>rd</sup> intron in modulating lineage specific expression of IRF-8.
<p>Schematic illustrations of the various BAC-IRF-8 constructs, to which a cassette containing a fluorescent reporter (mCherry) and a selectable marker (Neo driven by the PKG promoter) was inserted, are shown. (<b>A</b>) BAC-IRF-8.1- the cassette was inserted to the first ATG. (<b>B</b>) BAC-IRF-8.2- the cassette was swapped with the entire IRF-8 coding region (CDS). (<b>C</b>) The reporter construct BAC-IRF-8.3 is similar to that illustrated in panel A except that the 2<sup>nd</sup> intron was deleted. (<b>D</b>) The reporter construct BAC-IRF-8.4 is similar to that illustrated in panel A except that the 3<sup>rd</sup> intron was deleted. Exons (black boxes) are numbered. RAW and NIH3T3 cells were transfected with the various BAC constructs and the fluorescence intensity of the reporter gene in three different RAW and NIH3T3 stable clones, harboring 1–2 copies of the BAC reporter construct, was visualized under fluorescent microscope and quantified as described under Materials and Methods. (Students t-test,** p<0.01). Additionally, RNA was extracted from 3 independent clones for each BAC construct before and following treatment with IFN-γ and the Fold of Induction levels of the reporter gene and the endogenous IRF-8 were determined by real-time qRT-PCR (ii section of each panel).</p
