CRX Regulates RdCVF Figure 3. Mapping the active regions of the murine and human NXNL1 and NXNL2 promoter constructs using electroporation of mouse retinal explants. Retinal explants were electroporated at P0 with the GFP expression vector driven by different fragments of the Nxnl promoters and cultured for 5 days in vitro (DIV5). (A) NXNL1 (B) Nxnl1 (C) NXNL2 (D) Nxnl2. The intensity of fluorescence of a GFP reporter under the control of the chicken b-actin promoter (pCIG) is provided for comparison. doi:10.1371/journal.pone.0013075.g003 NXNL2 GFP reporter constructs. Fluorescence was monitored daily over a period of 8 days and the intensity of the signal was quantified (Figure 4). As expected, the intensity of RFP fluorescence increased over this time period, corresponding to the postnatal differentiation of the retina. The pattern of expression of the NXNL1 and NXNL2 promoters differed temporally from each other in that the NXNL1 constructs showed a continuous increase in activity with time whereas the NXNL2 constructs tended to show an early peak, usually at 1 day, and then show a decrease with time. The same regions that showed positive promoter activity in the single time-point studies, 2205 to 278 bp and 2351 to 278 bp for NXNL1 and Nxnl1, respectively, were found to be important for the observed temporal increase in promoter activity. Murine and human NXNL1 promoter constructs drive expression of GFP specifically in photoreceptors We next investigated the cell type specificity of the various promoter regions by determining the spatial distribution of GFP and RFP positive cells in slices of retinal explants 5 days after co- electroporation with the rhodopsin and NXNL1 and NXNL2 promoter constructs. With the human NXNL1 construct, 2205/ +57 GFP positive cells were observed exclusively in the outer nuclear layer, the part of the retina that corresponds to PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13075