CRX Regulates RdCVF Figure 2. Relative activity of murine, human NXNL1 and NXNL2 upstream region promoter fragments in HEK 293 and Y79 cells. (A) Human NXNL1 constructs. (B) Mouse Nxnl1 constructs. Sequence positions are numbered with the major putative TSS defined as +1. Luciferase values were normalized to control (pRL-TK) and the results are plotted as fold induction compared to pGL4.17 control plasmid. Values represent the average of at least three independent experiments, each done in triplicate. Error is shown as SEM. **p,0.01, ***p,0.001. The human constructs used in this and subsequent figures contain a C/T sequence variation at position -30 (T is the consensus sequence at this position) and an A/G sequence variation at position -8 (A is the consensus sequence at this position). The 277/+57 construct has a T at position -30 and a G at position -8; the 2205/+57 construct has a T at position -30 and G at position -8; the 2400/+57 construct has a T at position -30 and A at position -8; the 2501/+57 construct has a C at position -30 and A at position -8; the 21034/+57 construct has a T at position -30 and G at position -8; and the 22072/+57 construct has a C at position -30 and A at position -30. (C) Human NXNL2 constructs. (D) Mouse Nxnl2 constructs. Luciferase activity was normalized to control (pRL-TK) and the results are plotted as fold induction compared to pGL4.17 control plasmid. Values represent the average of at least three independent experiments done in triplicate. Error is shown as SEM. **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0013075.g002 to 2315) that presumably contains a critical DNA element(s). The intensity of GFP fluorescence was lower with the longer NXNL2 constructs (22033/+27 and 2519/+27), suggesting the presence of a negative element in the more far upstream region. Surprisingly, the murine Nxnl2 constructs tested with the mouse explants showed minimal and inconsistent activity, although it was clear that the 2195 to +70 region did have some activity (Figure 3D). We have previously reported that the temporal pattern of expression of RdCVF and RdCVF2 mRNA in the retina of the mouse shows increasing expression during development that then plateaus when the retina reaches its mature state, a pattern that is fairly similar to that of rhodopsin except that the actual level of expression is considerably lower [8,9]. As part of our character- ization of the various NXNL1 and NXNL2 promoter fragments, we performed a time course study with the retina explants, and also tested the bovine rhodopsin 2.2 kb promoter (22174/+70) as a comparative control [35]. A plasmid containing the 22174/+70 rhodopsin sequence upstream of red fluorescent protein (RFP) was co-electroporated with part of the deletion series of NXNL1 and PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13075