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Updated: May 10, 2026
Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice
Published on: July 11, 2016
A PCR-Based Method to Genotype Mice Knocked Out for All Four CD3 Subunits, the Standard Recipient Strain for
Alejandro Ferrer1, Adam G Schrum, Diana Gil
1Department of Immunology, College of Medicine, Mayo Clinic , Rochester, Minnesota.
Abstract:
The novel T-cell receptor (TCR)/CD3-retrogenic-reconstitution system represents a very useful strategy for studying TCR/CD3 signaling. Two retroviral vectors containing genes for all six subunits of the TCR/CD3 complex are used to transduce bone marrow precursors and reconstitute lethally irradiated recipient mice. Mice used in this system as bone marrow donors lack all four CD3 subunits (CD3γδɛζ(-/-)). These mice are generated by crossing the strains CD3ζ(-/-) and CD3γδɛ(-/-), the latter resulting from a knockout construct targeted to CD3ɛ that additionally silences the linked genes, CD3γ and CD3δ. Lacking mature T-cell function, CD3γδɛζ(-/-) mice are immunocompromised animals often produced by heterozygous breeding strategies on the C57BL/6 background. As a more rapid and reliable means to identify CD3γδɛζ(-/-) mice than previously described Northern and Southern blots, we designed polymerase chain reactions to distinguish knockout from wild-type CD3ɛ and CD3ζ alleles, facilitating the identification of CD3γδɛζ(-/-) mice.
Insights
A new retrogenic-reconstitution system aids T-cell receptor (TCR)/CD3 signaling studies. Novel PCR methods rapidly and reliably identify essential CD3γδɛζ(-/-) knockout mice for this research.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Studying T-cell receptor (TCR)/CD3 signaling is crucial for understanding immune responses.
- Existing methods for generating and identifying specific knockout mouse models can be time-consuming and unreliable.
- The CD3γδɛζ(-/-) mouse model, lacking all four CD3 subunits, is valuable for immunological research but requires efficient identification.
Purpose of the Study:
- To introduce a novel retrogenic-reconstitution system for studying TCR/CD3 signaling.
- To develop a rapid and reliable method for identifying CD3γδɛζ(-/-) knockout mice.
Main Methods:
- Utilized two retroviral vectors encoding all six subunits of the TCR/CD3 complex for bone marrow precursor transduction.
- Generated CD3γδɛζ(-/-) mice by crossing CD3ζ(-/-) and CD3γδɛ(-/-) strains.
- Designed specific polymerase chain reactions (PCR) to differentiate between knockout and wild-type CD3ɛ and CD3ζ alleles.
Main Results:
- Successfully established a novel TCR/CD3-retrogenic-reconstitution system.
- Developed PCR assays that provide a faster and more dependable method for identifying CD3γδɛζ(-/-) mice compared to traditional Northern and Southern blots.
- Facilitated the efficient identification of genetically modified mice crucial for TCR/CD3 signaling research.
Conclusions:
- The novel retrogenic-reconstitution system is a valuable tool for TCR/CD3 signaling research.
- The developed PCR strategy significantly improves the identification process for CD3γδɛζ(-/-) mice, enhancing experimental efficiency.
- This work provides a more robust approach for generating and characterizing essential mouse models in immunology.
