Knockout and re-expression system for mutant analysis in primary mouse T cells
4 affiliations
- 1Department of Molecular Immunology, Faculty of Biology, University of Freiburg
- 2Centre for Integrative Biological Signalling Studies (CIBSS), University of Freiburg
- 3Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg
- 4Nomis Center for Immunobiology and Microbial Pathogenesis & Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies
Abstract
We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo . This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3γ and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3γ-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3γ-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.
Related
- Clcn6 mRNA and ClC-6 protein expression in mouse central nervous system
- CRISPR/Cas9-mediated UBC knockout impairs CD44 expression and reduces the metastatic potential of 23132/87 gastric cancer cells
- Endothelial-specific Tie2 knockout mouse model - Tie2fl/fl;Cdh5-creERT2(+/-): a potent tool for inducible Tie2 deletion in vasculature across multiple organs.
- Version 2.0 of the Hermes transposon-based mutagenesis system for prototrophic mutant pool generation in Candida glabrata
- KH1 Domain of Poly C Binding Protein 1 (PCBP1) Interacts with Utrophin-A mRNA and Modulates Utrophin-A Expression in Mouse Myoblasts
BibTeX
@article{morfos2026knockout,
title = {{Knockout and re-expression system for mutant analysis in primary mouse T cells}},
author = {Vasileios Morfos and Meredith C. Frie and Daniel Peschkov and Juliana Wagner and Björn F. Lillemeier and Joanna Brzostek},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1946861},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1946861/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Knockout and re-expression system for mutant analysis in primary mouse T cells
AU - Vasileios Morfos
AU - Meredith C. Frie
AU - Daniel Peschkov
AU - Juliana Wagner
AU - Björn F. Lillemeier
AU - Joanna Brzostek
PY - 2026
DA - 2026/10/13
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1946861
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1946861/full
AB - We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo . This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3γ and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3γ-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3γ-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.
SN - 1664-3224
ER -