CRX Regulates RdCVF Figure 5. Cell-restricted expression of the murine and human NXNL1 and NXNL2 promoter constructs. Electroporated retinal explants were sectioned at DIV6, fixed and stained with antibodies against GFP (green, NXNL constructs) and RFP (red, rhodopsin construct marking rod photoreceptors). (A) NXNL1 (B) Nxnl1 (C) NXNL2 (D) Nxnl2. ONL: outer nuclear layer; INL: inner nuclear layer; and GCL: ganglion cell layer. doi:10.1371/journal.pone.0013075.g005 NXNL1 and NXNL2, respectively. We initially performed transient transfection analysis of a deletion series of murine and human Nxnl1 and Nxnl2 59-uptream region reporter constructs using both retinal (Y79 and 661W) cell and non-retinal (HEK 293 and COS- 1) cell lines. While promoter activity could be detected for a number of the constructs, the absence of evidence of cell-type specificity, i.e. absence of clear and consistent differences in promoter activity for the various constructs between the retinal and non-retinal cell lines, led us to utilize a more physiological assay for assessing retinal promoter activity. Although mouse transgenic studies are considered by many to be the gold standard for examining the cellular and temporal specificity of promoters, such studies are limited by position effects and the by the costs and time involved. As an intermediate approach that combines the practical advantages of cell line based approaches with the cellular specificity of transgenic studies, we adapted the electroporation of mouse retinal explants to provide rapid and reproducible quantitation of cell-specific promoter activity. Significantly, we PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13075