title: Rapid identification and expression of human TCRs in retrogenic mice.
abstract: 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.
authors: Department of Pediatrics, Section of Diabetes and Endocrinology, McNair Medical Institute, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA.
authors: Maran L
authors: ML
authors: Sprouse
authors: Benaroya Research Institute at Virginia Mason, Seattle, WA, USA.
authors: Gabriele
authors: G
authors: Blahnik
authors: Department of Pediatrics, Section of Diabetes and Endocrinology, McNair Medical Institute, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA.
authors: Thomas
authors: T
authors: Lee
authors: Department of Pediatrics, Section of Diabetes and Endocrinology, McNair Medical Institute, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA.
authors: Natalie
authors: N
authors: Tully
authors: Center for Human Immunobiology, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA.
authors: Pinaki
authors: P
authors: Banerjee
authors: Benaroya Research Institute at Virginia Mason, Seattle, WA, USA.
authors: Eddie A
authors: EA
authors: James
authors: Department of Pediatrics, Section of Diabetes and Endocrinology, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA.
authors: Maria J
authors: MJ
authors: Redondo
authors: Department of Pediatrics, Section of Diabetes and Endocrinology, McNair Medical Institute, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA.
authors: Matthew L
authors: ML
authors: Bettini
authors: Department of Pediatrics, Section of Diabetes and Endocrinology, McNair Medical Institute, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA. Electronic address: maria.bettini@bcm.edu.
authors: Maria
authors: M
authors: Bettini
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005838:Genotype; D057809:HEK293 Cells; D006680:HLA Antigens / Q000235:genetics / Q000276:immunology; D006801:Humans; D016688:Mice, Inbred NOD; D008822:Mice, Transgenic; D060885:Multiplex Polymerase Chain Reaction; D010641:Phenotype; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000276:immunology* / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D006680:HLA Antigens; D016693:Receptors, Antigen, T-Cell, alpha-beta
keywords: Human; Humanized mice; PCR; Single cell; T cell receptor; TCR