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Identification of the transgenic integration site in 2C T cell receptor transgenic mice

Chae-Yeon Son¹, Brian B Haines^2,3, Andreas Luch^2,4

¹Department of Integrative Bioscience and Biotechnology, Institute of Anticancer Medicine Development, Sejong University, Seoul, Korea.

Transgenic Research

|August 23, 2018

Abstract:

2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vβ13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27 weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3' region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.

Insights

The 2C T cell receptor (TCR) transgene integration site was identified on chromosome 1, near the cytosolic phospholipase A2 group IVA (cPLA2) gene. This finding is crucial for understanding T cell lymphoma development in 2C transgenic mice.

Area of Science:

Immunology
Genetics
Molecular Biology

Background:

2C T cell receptor (TCR) transgenic mice are vital tools for studying T cell receptor interactions and cytotoxic T lymphocyte (CTL) mechanisms.
Previous studies in mutant mice (P13) lacking the Vβ13 promoter showed accelerated T cell lymphoma when engineered with the 2C transgene.

Purpose of the Study:

To identify the integration site of the 2C TCR transgene in mice.
To investigate the relationship between transgene integration and T cell lymphoma development.
To determine the expression levels of nearby genes, specifically cPLA2, in relation to lymphoma.

Main Methods:

Southern blotting was used to detect 2C-specific DNA fragments.
Digestion-circularization-polymerase chain reaction (DC-PCR) with inverse primers amplified the integration junction.
Sequence analysis identified the integration site and adjacent genes.
cPLA2 gene expression was analyzed in thymi, lymph nodes, and lymphomas using quantitative methods.

Main Results:

The 2C TCR α transgene was confirmed to integrate into chromosome 1.
The cytosolic phospholipase A2 group IVA (cPLA2) gene was identified as the nearest gene to the integration site.
cPLA2 expression was significantly upregulated in the thymi and T cell lymphomas of 2C transgenic mice.
No alteration in cPLA2 expression was observed in the lymph nodes of these mice.

Conclusions:

This study reports the precise integration site of the 2C TCR transgene for the first time.
The proximity of the transgene to cPLA2 and its subsequent upregulation suggest a role in accelerated T cell lymphoma.
Understanding the integration site and its genetic consequences will improve the utility of 2C transgenic models in immunological research.

Read full analysis

Keywords:

2C TCR transgenic mice DC-PCR Integration site T cell lymphoma cPLA2

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