a practical approach to t-cell receptor cloning and expression一个实际的t细胞受体的克隆和表达方法.pdfVIP

A Practical Approach to T-Cell Receptor Cloning and Expression ´ ¨ 1 ˚ 2,3 1 ˚ 1¤ 1 Sebastien Walchli *, Geir Age Løset , Shraddha Kumari , Jorunn Nergard Johansen , Weiwen Yang , Inger Sandlie2,3, Johanna Olweus 1,4* 1 Department of Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway, 2 Department of Molecular Biosciences and Centre for Immune Regulation, University of Oslo, Oslo, Norway, 3 Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway, 4 Institute of Clinical Medicine, University of Oslo, Oslo, Norway Abstract Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 59-RACE amplification. We here present an improved 59-RACE protocol that represents a fast and reliable way to identify a TcR from 105 cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed pr