as compared to 228 bp in the retina, suggesting a difference in the mechanism controlling its expression in photoreceptor versus HeLa cells. Murine and human NXNL1 and NXNL2 promoter activity in retinal and non-retinal cell lines In most instances, the major DNA elements regulating promoter activity are located in the 59-upstream region of a given gene [28–30]. In order to map the promoter regions of the murine and human NXNL1 and NXNL2 genes, we used PCR to construct a collection of reporter plasmids containing various sequences 59 to the respective gene’s ATG site into the promoterless luciferase reporter vector pGL4.17. The human samples were generated from DNA that was derived from a single donor’s white blood cells. DNA sequencing of the resultant constructs, as well as sequencing of additional independent PCR products, and comparison with the UCSC database revealed both previously identified and novel sequence variants within the upstream regions of both the NXNL1 and NXNL2 genes (since the term ‘‘polymorphism’’ is generally used to refer to sequence variants that occur at a frequency of at least 1% in a population, and we do not have any frequency data on the variants we identified, in the text below we will refer to these as ‘‘sequence variants’’). The identity and location of the detected sequence variants and their occurrence within the various reporter constructs studied are noted in the appropriate figure legends. For initial analysis, reporter plasmids were transfected into the Y79 retinoblastoma cell line, which based on the NCBI EST database expresses RdCVF, and into the non-retinal cell line HEK 293, which was derived from adenoviral DNA transformed human embryonic kidney cells [31]. The activity of the various promoter constructs was measured using a dual luciferase assay to normalize expression relative to that of a HSV-thymidine kinase promoter (pRL-TK- renilla), and the data are presented as fold induction compared to the empty pGL4.17 plasmid. With the NXNL1 transfection series and Y79 retinoblastoma cells, we found similar levels of reporter activity with the 22072 to +57 bp, 21034 to +57 bp, and 2501 to +57 bp constructs (Figure 2A). In HEK 293 cells we found similar levels of reporter activity with the 21034 to +57 bp and 2501 to +57 bp constructs, but observed significantly less activity with the 22072 to +57 bp construct, suggesting that a negative DNA element(s) active in 293 cells, but not in the retina, might be present in the 22072 to 21034 bp region. Using an analogous approach with a set of shorter deletion constructs, we localized much of the promoter activity to the immediate upstream sequence in Y79 cells, and probably also HEK293 cells, to the region between 2205 and 277 bp (Figure 2A). Here again, there is a suggestion of a negative regulatory element specific to HEK 293 cells, this time localized to the 2400 to 2205 bp region. In contrast to these findings of significant promoter activity with the human NXNL1 upstream region, with murine Nxnl1 we identified only minimal promoter activity with the constructs tested (Figure 2B). Similar results were obtained with two other cell lines, the mouse cone-like line 661W [32] cells and COS-1 cells, derived from African green monkey kidney cells (Figure S1). As noted above, there is surprisingly little sequence conservation between the murine and human NXNL1 upstream regions. Thus one possible explanation for the mouse constructs lack of significant promoter activity with the monkey and human cells is that they may lack the transcription factors necessary to recognize the murine regulatory elements, and based on the results with mouse retinal explants (see below), this does seem likely to be at least part of the explanation (Figure 2B). However, species CRX Regulates RdCVF difference does not explain the difference observed with the murine 661W cells. In the case of the NXNL2 gene, contrary to that observed with NXNL1, relative promoter activity was higher in the non- photoreceptor cell line HEK 293 than in Y79 cells (Figure 2C). The major component of the observed positive regulatory activity, in both HEK 293 and Y79, cells was localized to the region between 2393 and 2315 bp (Figure 2C). As with the human gene, the murine Nxnl2 promoter constructs were also considerably more active in HEK 293 than in Y79 cells (Figure 2D). The step-wise reduction in activity observed with the shorter deletion constructs in the HEK 293 cells suggests the presence of multiple regulatory elements, each of moderate activity (Figure 2D). Comparable results were obtained with 661W and COS-1 cells (Figure S1). Murine and human NXNL1 and NXNL2 promoter activity in electroporated mouse retinal explants Based on the lack of clear retinal specificity in the above described cell line studies, which in fact was not surprising given the clear importance of cellular environment on transcriptional activity, we switched to electroporation of perinatal mouse retinal explants, an ex vivo approach that we thought would be more amenable to analysis of retinal cell-specific promoter elements. Prior studies by Matsuda and Cepko had shown that mouse retina can be transfected reasonably efficiently by electroporation and that in these experiments retinal promoters tend to retain their cell type specificity [33,34]. Using an adaptation of their methods, we performed electroporation with various green fluorescent protein (GFP) reporter constructs analogous to the previously described Nxnl1 and Nxnl2 luciferase constructs, and then quantified the activity of the promoter fragments by measuring the cellular fluorescence of the electroporated retinas. Mouse retinal explants were electroporated at post-natal day 0 (P0) and maintained in culture 5 days. Fluorescent signals were captured with a Charge Coupled Device (CCD) camera. Using this approach, although there was certainly significant variation from electroporation to electroporation, even with the same construct, we were able to observe fluorescent signals whose intensity was highly dependent upon the upstream sequence contained in the reporter construct. For example, with human NXNL1, we observed fairly similar expression levels with the 22072 to +57 bp, 2501 to +57 bp, and 2205 to +57 bp constructs, but when the upstream region was deleted to 277 to +57 bp there was essentially complete loss of activity (Figure 3A). Although consistent in a general sense with the pattern of the Y79 and HEK 293 results, quantitatively the mouse retina result was significantly different in that with the cell lines the decrease in activity with deletion of the 2205 to 277 bp region resulted in only a 40% decrease in activity (Figure 2A). Other differences between the cell line and retinal explant results were even more striking. Unlike with the cell lines, in the mouse explants the murine Nxnl1 promoter constructs are more active than the human NXNL1 (compare Figure 2A-B with Figure 3A-B). With the cell lines, the Nxnl1 construct with the maximal activity was the 277 to +51 bp fragment (Figure 2B), whereas this construct had no detectable activity in the explant system (Figure 3B). The consistently low activity of the 21749/+51 bp construct in the explant system compared to the 2924 to +51 bp construct most probably indicates the presence of a negative regulatory element located between 21749 and 2924 bp. With the NXNL2 promoter constructs, the retinal explant results were fairly consistent with those obtained with the cell lines (compare Figure 2C and Figure 3C). As with the cell lines, the sequence from 2393 to 2315 bp was required for significant promoter activity. This finding delineates a region of 79 bp (2393 PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13075