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A P2rx7 Passenger Mutation Affects the Vitality and Function of T cells in Congenic Mice
Marco Er-Lukowiak1, Yinghui Duan1, Francois Rassendren2,3
1Department of Neurology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg-Eppendorf, Germany.
Abstract:
Among laboratory mouse strains many genes are differentially expressed in the same cell population. As consequence, gene targeting in 129-derived embryonic stem cells (ESCs) and backcrossing the modified mice onto the C57BL/6 background can introduce passenger mutations in the close proximity of the targeted gene. Here, we demonstrate that several transgenic mice carry a P2rx7 passenger mutation that affects the function of T cells. By the example of P2rx4tm1Rass we demonstrate that P2X4ko T cells express higher levels of P2X7 and are more sensitive toward the P2X7 activators ATP and NAD+, rendering these cells more vulnerable toward NAD-induced cell death (NICD) compared with wild type (WT). The enhanced NICD sensitivity confounded functional assays e.g. cytokine production and cell migration. Our results need to be considered when working with P2rx4tm1Rass mice or other 129-based transgenic strains that target P2rx7 neighboring genes.
Insights
Passenger mutations near targeted genes in mice can affect T-cell function. Specifically, a P2rx7 mutation in P2X4 knockout mice increases sensitivity to cell death, impacting experimental results.
Area of Science:
- Immunology
- Genetics
- Molecular Biology
Background:
- Gene targeting in laboratory mice often involves embryonic stem cells (ESCs) from the 129 strain, followed by backcrossing onto the C57BL/6 background.
- This process can lead to passenger mutations in genes located near the intended target, potentially confounding experimental outcomes.
- Differential gene expression between mouse strains can exacerbate the issue of unintended genetic alterations.
Purpose of the Study:
- To investigate the impact of passenger mutations on T-cell function in genetically modified mice.
- To characterize the effects of a specific passenger mutation in the P2rx7 gene.
- To highlight the implications of these findings for interpreting functional assays in transgenic mouse models.
Main Methods:
- Analysis of gene expression in T-cells from wild-type (WT) and genetically modified mice.
- Assessment of P2X7 receptor expression levels.
- Functional assays measuring T-cell sensitivity to P2X7 activators like ATP and NAD+.
- Evaluation of NAD-induced cell death (NICD) and its impact on cellular functions such as cytokine production and migration.
Main Results:
- Transgenic mice carrying a P2rx7 passenger mutation were identified.
- T-cells from P2rx4 knockout mice (P2X4ko) exhibited elevated P2X7 receptor expression.
- P2X4ko T-cells demonstrated increased sensitivity to P2X7 activators (ATP and NAD+).
- These cells were more vulnerable to NAD-induced cell death (NICD) compared to WT cells.
- The heightened NICD sensitivity interfered with the accurate assessment of T-cell functions, including cytokine production and cell migration.
Conclusions:
- Passenger mutations, particularly in the P2rx7 gene, can significantly alter T-cell function and experimental readouts.
- The enhanced sensitivity to NICD in P2rx4tm1Rass mice confounds functional assays, necessitating careful consideration.
- Researchers using 129-based transgenic strains, especially those targeting genes near P2rx7, must account for potential passenger mutation effects.
