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BACKGROUND: Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming. RESULTS: Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA+ T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA+ cells is achievable when these modifications are used in conjunction. CONCLUSION: The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods.

Original publication

DOI

10.1186/1472-6750-13-98

Type

Journal article

Journal

BMC Biotechnol

Publication Date

12/11/2013

Volume

13

Keywords

Antigens, CD45, Butyric Acid, Calcium Phosphates, Gene Transfer Techniques, Genetic Vectors, HEK293 Cells, Humans, Lentivirus, Lipids, Plasmids, T-Lymphocytes, Transduction, Genetic, Transfection, Transgenes