CRX Regulates RdCVF Figure 8. The proximal CRX binding element within the NXNL1 is important for photoreceptor cell type specificity. P0 mouse retinas were electroporated with the rhodopsin RFP expression construct and the indicated GFP construct. (A) Comparison of 79+134 wild-type (wt) and 79+134 CRX mutant constructs (mut). The 79+134 wt and CRX mutant constructs both have a C at position -30 and a G at position -8. (B) Quantified fluorescence intensity of the electroporated retinal explants. (C) Sectioned retinal explants showing the regional localization of reporter expression in the layers of the retina. ONL: outer nuclear layer and INL: inner nuclear layer. ***p,0.001. doi:10.1371/journal.pone.0013075.g008 specificity within the retina. Immortalized cell lines are appropri- ate and provide a powerful system to reveal potent cis-acting elements with broad cellular spectrum, and also can be useful for gain of function studies in which non-expressed cell type-specific trans-acting factors are added to the cells, but are of limited use for revealing the promoter elements responsible for conferring highly cell-restricted patterns of expression. The deletion series of the NXNL1 promoter constructs revealed a critical region proximal to the transcriptional start site of both the human (2205 to 277 bp) and mouse (2351 to 277 bp) genes (Figure 3A and B). Within these regions, cis elements are able to confer promoter activity to the reporter constructs, and within the context of more downstream sequences to restrict expression to the naturally RdCVF expressing cells, photoreceptor cells and, to a lesser extent, bipolar cells (Figure 5A and B). The promoter activity scored for these constructs (NXNL1 2205/+87 and Nxnl1 2351/+51) seems to depend on the state of differentiation of the retinal cells, and mostly of the differentiation state of photorecep- tors since the activity gradually increases in concert with the activity of the rhodopsin promoter construct with time (Figure 4). PLoS ONE | www.plosone.org 12 October 2010 | Volume 5 | Issue 10 | e13075