Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells
1 Laboratory of Molecular and Cellular Biology, Faculty of Science and Engineering, Graduate School, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan
2 Graduate School of Innovative Life Science, University of Toyama, Toyama-shi, Toyama, 930-8555, Japan
3 Laboratory of Molecular and Cellular Biology, Faculty of Science and Engineering, Graduate School, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan
BMC Biotechnology 2011, 11:39 doi:10.1186/1472-6750-11-39Published: 13 April 2011
Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.
We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells.
The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.