streamlined-single-cell-tcr-isolation-generation-retroviral-vectors

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for <em>In Vitro </em>and <em>In Vivo</em> Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Baylor College of Medicine, Texas Children's Hospital

Single-cell paired TCR identification is a powerful tool, but has been limited in its previous incompatibility with further functional analysis. The current protocol describes a method to clone and functionally evaluate in vivo TCRs derived from single antigen-responsive human T cells and monoclonal T cell lines. We have improved upon current PCR-based TCR sequencing protocols by developing primers that allow amplification of human TCRα and TCRβ variable regions, while incorporating specific restriction cut sites for direct subcloning into the template retroviral vector. This streamlined approach for generating human:mouse chimeric TCR vectors allows for rapid TCR expression in humanized-retrogenic (hu-Rg) mice through retroviral mediated stem cell gene transfer. Using widely available techniques and equipment, this method is easily adaptable by most laboratories. This is the first TCR identification protocol that is efficiently combined with subsequent in vivo TCR expression.

Natalie Tully

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