Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Abstract

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCRα and TCRβ chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR. Video LinkThe video component of this article can be found at

Ectopic Expression of Self-Antigen Drives Regulatory T Cell Development and Not Deletion of Autoimmune T Cells

“…Transient transfection of HEK-293T cells was performed as previously described (38-40). Briefly, cells were cultured in 6-well plates at 2×10 5 cells/well overnight at 37°C.…”

“…Retroviral transduction of murine bone marrow cells was performed as previously described (38-41). Transduced bone marrow cells were collected, washed and resuspended in PBS with 0.5% FBS.…”

“…Additionally, higher affinity interactions with self-peptide/MHC and TCR have been shown to be required for the expression and maintenance of Foxp3 (35-37). In order to address the impact of thymic antigen dose and pMHC stability on the selection of insulin specific T cells, we utilized TCR retrogenic technology (38-40) to generate mice that co-express either low or high affinity insulin reactive TCRs (on T cells) and either insulin or insulin mimetope R22E (on antigen presenting cells (APCs)). Our studies reveal that ectopic insulin antigen expression during thymocyte development of high or low affinity insulin reactive TCRs does not result in the deletion of these T cells, however all mice were protected from developing T1D long term.…”

Rapid identification and expression of human TCRs in retrogenic mice

“…This vector contains a fluorescent reporter cassette (Ametrine: ex.406 em.526) to facilitate the identification of transduced cells, and is compatible with the widely used GFP reporter. The original mouse 8-1.1 TCR-pMSCVII-Ametrine (pMIA) vector (Bettini et al, 2014; Lee et al, 2016) was modified by quick change mutagenesis to incorporate the Sna bI, Sac II, Bst bI, and Mfe I restriction enzyme cut sites on either side of the TCR α and β variable regions of the encoded ‘placeholder’ TCR.…”

“…Humanized-retrogenic mice were generated in a manner similar to that previously described for mouse TCR retrogenic mice (Bettini et al, 2013; Lee et al, 2016). Bone marrow was harvested from the femur, tibia, hip, and humerus of 6–12 week old donor mice 72 h after intraperitoneal injection of 150 mg/kg 5-fluoruracil (5FU) in PBS at a ratio of 1.5 donors: 1 recipient.…”