A High-Throughput Strategy for T-Cell Receptor Cloning and Expression

Qiong Xia; Huang Huang; Mark M Davis

doi:10.1007/978-1-0716-2712-9_12

A High-Throughput Strategy for T-Cell Receptor Cloning and Expression

Methods Mol Biol. 2022:2574:251-264. doi: 10.1007/978-1-0716-2712-9_12.

Authors

Qiong Xia¹, Huang Huang¹, Mark M Davis^{2

3

4}

Affiliations

¹ Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA.
² Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA. mmdavis@stanford.edu.
³ Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. mmdavis@stanford.edu.
⁴ The Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. mmdavis@stanford.edu.

Abstract

Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics.

Keywords: Lentivirus package; TCR expression; TCR high-throughput cloning; Transfection.

MeSH terms

Cloning, Molecular
Genes, T-Cell Receptor*
Humans
Receptors, Antigen, T-Cell* / genetics
Receptors, Antigen, T-Cell* / metabolism

Substances

Receptors, Antigen, T-Cell

Abstract

MeSH terms

Substances

Grants and funding