PMID: 11933214Apr 5, 2002Paper

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein

The Journal of Gene Medicine
Alexander E AnnenkovYuti Chernajovsky

Abstract

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. 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. 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. W...Continue Reading

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Citations

Jan 28, 2004·Proceedings of the National Academy of Sciences of the United States of America·Joanna M ClarkAndrew P Cope
Jul 6, 2010·Chinese Medical Sciences Journal = Chung-kuo I Hsüeh K'o Hsüeh Tsa Chih·Feng ChenChih-chuan Liang
Feb 6, 2008·Journal of Virological Methods·E L BeaudoinR P Junghans

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