Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells.
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.


CONCLUSIONS
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.
Kennedy Institute of Rheumatology, Roosevelt Drive, Headington OX3 7FY, Oxford, UK. adam.cribbs@kennedy.ox.ac.uk.
Adam P
AP
Cribbs
Alan
A
Kennedy
Bernard
B
Gregory
Fionula M
FM
Brennan
D020148:Butyric Acid / Q000378:metabolism; D002130:Calcium Phosphates / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008055:Lipids / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection; D019076:Transgenes
D002130:Calcium Phosphates; D008055:Lipids; C086724:Lipofectamine; D020148:Butyric Acid; C020243:calcium phosphate; D017493:Leukocyte Common Antigens