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Related Experiment Video

Updated: Aug 29, 2025

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs
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A High-Throughput Strategy for T-Cell Receptor Cloning and Expression.

Qiong Xia1, Huang Huang1, Mark M Davis2,3,4

  • 1Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|September 10, 2022
PubMed
Summary

A new recombination-based cloning protocol simplifies T-cell receptor (TCR) gene cloning and expression. This method enables rapid TCR characterization and supports the development of TCR-based cancer immunotherapies.

Keywords:
Lentivirus packageTCR expressionTCR high-throughput cloningTransfection

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Area of Science:

  • Molecular Biology
  • Immunology
  • Biotechnology

Background:

  • T-cell receptor (TCR) gene expression is crucial for TCR characterization and epitope identification.
  • The growing interest in TCR-based cancer immunotherapy necessitates efficient methods for TCR cloning and expression.

Purpose of the Study:

  • To develop a simple, rapid, and robust method for cloning and expressing T-cell receptor (TCR) transgenes.
  • To facilitate TCR characterization and support the development of TCR-mediated therapeutics.

Main Methods:

  • Construction of all human TRAV and TRBV genes into individual plasmids.
  • Utilizing Golden Gate cloning to ligate short CDR3 fragments to corresponding V gene plasmids.
  • Employing a recombination-based cloning protocol for transgene transfer into expression systems.

Main Results:

  • The protocol enables simple and rapid transfer of TCR transgenes into various expression systems.
  • Significantly improved efficiency in individual TCR cloning and mutagenesis.
  • Established a flexible, high-throughput method for TCR analysis.

Conclusions:

  • The developed recombination-based cloning protocol offers an efficient strategy for TCR cloning and expression.
  • This method advances TCR analysis and supports the development of TCR-mediated therapeutics for cancer immunotherapy.