Abstract
The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases. P2X7 effects were also described in neurons and astrocytes but its localisation and function in these cell types has been challenging to demonstrate in situ. BAC transgenic mouse lines have greatly advanced neuroscience research and two BAC-transgenic P2X7 reporter mouse models exist in which either a soluble EGFP (sEGFP) or an EGFP-tagged P2X7 receptor (P2X7-EGFP) is expressed under the control of a BAC-derived P2rx7 promoter. Here we evaluate both mouse models and find striking differences in both P2X expression levels and EGFP reporter expression patterns. Most remarkably, the sEGFP model overexpresses a P2X4 passenger gene and sEGFP shows clear neuronal localisation but appears to be absent in microglia. Preliminary functional analysis in a status epilepticus model suggests functional consequences of the observed P2X receptor overexpression. In summary, an aberrant EGFP reporter pattern and possible effects of P2X4 and/or P2X7 protein overexpression need to be considered when working with this model. We further discuss reasons for the observed differences and possible caveats in BAC transgenic approaches.
Highlights
The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases
Stability and regulation of the resulting reporter mRNA and protein might differ from that of the gene of interest, (ii) critical regulatory elements may be absent in the chosen bacterial artificial chromosome (BAC), (iii) possible position effects caused by the random integration of the modified BAC in the genome should be excluded by comparison of several transgenic mouse lines, and (iv) integration of multiple fluorescent reporter gene copies is usually required to allow efficient visualization
We further demonstrate that the expression pattern of soluble enhanced green fluorescent protein (EGFP) can be reconciled with partial neuronal expression while the P2X7-EGFP fusion protein is dominantly expressed in microglia and oligodendrocytes but not detectable in neurons
Summary
The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases. To allow cell type-specific visualization and analysis of protein expression in complex tissues, the generation of bacterial artificial chromosome (BAC) transgenic reporter mice has proven to represent an invaluable method that greatly advanced neuroscience research[13,14,15]. In this approach, reporter proteins, such as enhanced green fluorescent protein (EGFP), are either directly expressed under the control of BAC-derived regulatory sequences of the gene of interest or a BAC-controlled Cre recombinase is used to cell- drive fluorescent protein expression in floxed reporter mouse lines. The EGFP sequence was integrated in frame into the last exon of the P2rx[7] gene resulting in the expression of a P2X7-EGFP fusion p rotein[24]
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