Deviant reporter expression and P2X4 passenger gene overexpression in the soluble EGFP BAC transgenic P2X7 reporter mouse model

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.

Microglial Activation Modulated by P2X4R in Ischemia and Repercussions in Alzheimer’s Disease

“…In addition, it has been reported P2X4R negative mice with increased P2X7R expression with passenger mutations altering the receptor’s function ( Er-Lukowiak et al, 2020 ; Ellegaard et al, 2021 ). Similar results were obtained using a transgenic mouse line that expresses soluble GFP by P2X7R promoter, increasing the expression of P2X4R ( Ramírez-Fernández et al, 2020 ). However, has been reported a concomitant decrease in the expression of P2X4R and 7 in liver and kidney of mice deficient for P2X4R or 7 ( Craigie et al, 2013 ; Besnard et al, 2016 ).…”

Astrocytic and Oligodendrocytic P2X7 Receptors Determine Neuronal Functions in the CNS

“…Although in both P2X7 reporter models the EGFP constructs are expected to be expressed under the control of the P2rx7 promoter, there were substantial differences in their cell-type-specific expression, and serious concerns were raised regarding the reliability of the reporter expression in the sEGFP mouse (Ramirez-Fernandez et al, 2020 ). For example, the sEGFP model overexpresses a P2X4 passenger gene, and sEGFP shows clear neuronal localization, but appears to be absent in microglia.…”

Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

“…The qPCR results from mRNA demonstrated that the overexpression of Cdx1 is essentially derived only from exons 2 and 3 of Cdx1 ( Figure 4 B), indicating that Cdx1 overexpression is derived from the BAC transgene. An increase in the gene copy number generally results in upregulated expression, sometimes disproportionately higher in a tandem arrangement ( Loehlin and Carroll, 2016 ), and the “passenger genes” on BAC clones are known to be upregulated in BAC transgenic mice ( Farrar et al., 2021 ; Gong et al., 2010 ; Ramirez-Fernandez et al., 2020 ). In addition, considering the fact that the upregulation of Slc6a7 , Arsi , and Cdx1 unnaturally stands out in terms of fold change and reliability compared to more mild and variable changes in other genes in RNA-seq analysis ( Figure S3 B and Table S1 ), it is highly likely that a significant proportion of these upregulated genes is the result of multiplied copy number of genes from the integrated BAC transgene.…”