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Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus
Alexander E Annenkov1, Gordon M Daly, Yuti Chernajovsky
1Bone and Joint Research Unit, St Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary, University of London, Charterhouse Square, London EC1M 6BQ, UK.
Background:
Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication-deficient retrovirus can be used in immunological studies and for cell-based gene therapy.
Methods:
Packaging cells GP+E86 producing replication-deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2gamma or C2xi, were generated by calcium phosphate-mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T-cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe.
Results:
PT67 cells produced approximately 50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them for retroviral RNA content), the transduction efficiency of mouse T-cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two- to three-fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4(+) T cells and up to 80% of B cells could be transduced with a transgene in replication-deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol.
Conclusions:
This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes.