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Deviant reporter expression and P2X4 passenger gene overexpression in the soluble EGFP BAC transgenic P2X7 reporter
Antonio Ramírez-Fernández1, Lidia Urbina-Treviño2, Giorgia Conte3
1Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, LMU Munich, Munich, Germany.
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.
Insights
Two BAC transgenic mouse models for the P2X7 receptor show significant differences in reporter gene expression and potential overexpression of P2X4 and P2X7, impacting their use in brain disease research.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- The P2X7 receptor, a key player in microglia and brain diseases, has challenging in situ localization and function in neurons and astrocytes.
- BAC transgenic mouse models offer advanced tools for neuroscience research, with two existing P2X7 reporter models: sEGFP and P2X7-EGFP.
Purpose of the Study:
- To evaluate and compare two BAC-transgenic P2X7 reporter mouse models.
- To identify differences in P2X expression levels and EGFP reporter patterns.
- To assess potential functional consequences of observed P2X receptor overexpression.
Main Methods:
- Evaluation of two BAC-transgenic P2X7 reporter mouse models (sEGFP and P2X7-EGFP).
- Analysis of P2X expression levels and EGFP reporter expression patterns.
- Preliminary functional analysis in a status epilepticus model.
Main Results:
- Striking differences observed in P2X expression levels and EGFP reporter patterns between the two models.
- The sEGFP model exhibits P2X4 passenger gene overexpression and neuronal, but not microglial, sEGFP localization.
- Preliminary data suggest functional consequences of P2X receptor overexpression in a status epilepticus model.
Conclusions:
- Aberrant EGFP reporter patterns and potential P2X4/P2X7 overexpression in the sEGFP model require careful consideration.
- Differences in BAC transgenic approaches can lead to significant variations in reporter gene expression and receptor localization.
- Further investigation into the caveats of BAC transgenic approaches is warranted for accurate interpretation of P2X7 receptor studies.
