Generation of T Cell Receptor Retrogenic Mice

Abstract

The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus‐mediated stem cell–based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector‐encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

Single-cell lineage mapping of a diverse virus-specific naive CD4 T cell repertoire

“…Theoretically, a few lineage-specific motifs identified in this study could exhibit altered affinity and/or dwell time for their cognate GP 66-77 -I-A b peptide MHC complex, which may in turn affect TCR signaling and cell fate decisions ( Tubo et al, 2013 ). Detailed delineation of these possibilities will be needed in future studies using TCR retrogenic mice ( Holst et al, 2006 ; Kong et al, 2019 ). However, TCR structure is only one of many factors impacting CD4 T cell fate determination, which is also heavily influenced by well-established external factors during infection ( Choi et al, 2011 ; Pepper et al, 2011 ).…”

Increased TCR signaling in regulatory T cells is disengaged from TCR affinity

“…To determine antigen specificities and relative antigen reactivity of islet infiltrating CD4+ effector and Foxp3+ regulatory T cells, 14 TCRs (7 from Teffs and 7 from Tregs) were cloned directly from infiltrated islets of a prediabetic NOD.Foxp3-GFP female mouse (Table 1). T cells expressing isolated TCRs were generated using TCR retrogenic approach (TCR Rg) as previously described 19,20 . T cells isolated from spleens of single TCR Rg mice were expanded in vitro and stimulated with the two dominant CD4 epitopes targeted in NOD diabetes: insulin B:9-23 and chromogranin/insulin hybrid peptide 2.5HIP [21][22][23] .…”

“…TCR retrogenic mice were generated as previously described 19 . Briefly, 5-fluorouracil (NDC, 63323-117-51) was given intraperitoneally to bone marrow donor mice (0.15mg/g body weight).…”