Figure 1
Comparison of BAC transgenic P2X7 reporter mice and P2rx4 and P2rx7 expression in these mice. (A) The sEGFP sequence followed by a poly(A) signal was inserted in the start ATG in exon 1 of the P2rx7 gene. The recombination strategy resulted in co-integration of the pLD53.SC2 vector downstream of the poly(A) signal. In case of the P2X7-EGFP fusion construct, a Strep-His-linker followed by the EGFP sequence was inserted in front of the Stop TGA in exon 13 of P2rx7. Use of two homology arms prevented vector integration. Intraflagellar transport protein Ift81 and, in case of the sEGFP model, P2rx4 are introduced as passenger genes together with P2rx7. In the P2X7-EGFP model, Ift81 is destroyed by Sac II linearization. HomA/B: gene-specific homology arms; opened grey circle: vector backbone of the BAC. For further details see Table 1. (B) Quantitative RT-PCR analysis of hippocampal P2rx4 and P2rx7 expression in both mouse models compared to wt mice. Data were normalized to expression of β-actin and the respective levels in wt controls. Bars represent mean ± SEM from 2 independent experiments and 6–7 mice (both sexes, around eight weeks of age). Significance was analysed using the Mann–Whitney test. **p < 0.01. (C) Representative Western blot analysis of P2X4 and P2X7 protein expression. 50 µg of total lung protein (extracted in 1% NP-40) were loaded per lane. Proteins were immunoblotted and quantified by infrared imaging with fluorescent secondary antibodies. Bars represent mean ± SEM from two independent experiments and 6 mice (both sexes, 10–35 weeks of age). Significance was analysed using the Mann–Whitney test. **p < 0.01. Note that the overexpression of P2X4 leads to aggregation and multimer formation in the sEGFP sample. The full gels are shown in Supplementary Fig. 6. (D) Immunofluorescence staining with a P2X4 antibody to demonstrate increased P2X4 expression level in the sEGFP reporter mouse. Three different animals were analysed per group, and representative images of cerebellum and hippocampus are shown. GL, granular cell layer; ML, molecular cell layer; DG, dentate gyrus; PCL, Purkinje cell layer; WM, white matter; CA3, cornu amonis region 3; Scale bars 100 µm.
