Lentivirus Transduction of Hematopoietic Cells | |
Ming-Jie Li and John J. Rossi Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010 Excerpted from Gene Transfer: Delivery and Expression of DNA and RNA Edited by Theodore Friedmann and John Rossi |
ABSTRACT |
Efficient transfer and sustained expression of transgenes are among the most important issues in gene delivery technologies. The majority of hematopoietic cells, including hematopoietic stem/progenitor cells and terminally differentiated cells, such as primary T cells and macro phages, are nondividing or slowly self-renewing. These cell types are refractory to most nonviral or retroviral delivery methods. Lentiviral vectors are capable of transducing nondividing cells and maintaining long-term and sustained expression of the transgenes. Many hematopoietic cell types have been successfully transduced with lentiviral vectors carrying a variety of genes. Lentiviral vectors are becoming useful for many delivery protocols, such as long-term expression of short hairpin RNA (shRNA) and functional genetics. They may also have great potential in gene therapy. This protocol describes lentivirus-vector-based delivery of foreign genes to hematopoietic cells. The method is applicable to various cell types in experiments that require long-term transgene expression. |
MATERIALS |
Reagents |
|
Equipment |
|
METHOD |
|
TROUBLESHOOTING |
Problem (Step 20): Some cell types, such as CEM (a human T cell line), are difficult to transduce by the standard protocol. Solution: For those cell types that resist transduction by normal means, centrifugation can substantially enhance the transduction efficiency. Treat these cells as follows. Combine 2 x 105 cells in 1 ml of culture medium in a 15-ml centrifuge tube with vector and 4 µg/ml polybrene. Centrifuge at 900g for 30 minutes at 20°C. Then, without removing the supernatant, use a pipette to resuspend the cell pellet and transfer the cells to the culture plate. Incubate the cells overnight, replace the medium, and continue the culture. |
ACKNOWLEDGMENTS |
We thank Dr. Jiing-Kaun Yee for providing pHIV7-GFP and the packaging plasmids. The authors were supported by National Institutes of Health grants AI29329, AI42552, and HL074704. |
Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult: Gene Transfer: Delivery and Expression of DNA and RNA, A Laboratory Manual, edited by Theodore Friedmann and John Rossi, © 2006 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 75-82. |
Copyright © 2006 by Cold Spring Harbor Laboratory Press. All rights reserved. No part of these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction modification, storage in a retrieval system or retransmission, in any form or by any means, electronic, mechanical, or otherwise, for reasons other than personal use, is strictly prohibited without prior written permission. |