Protocol abstract
Nature Protocols 1, - 2688 - 2700 (2007)
Published online: 11 January 2007 | doi:10.1038/nprot.2006.409
Subject Categories: Immunological techniques | Microbiology and virology | Model organisms
Use of a lentivirus/VSV pseudotype virus for highly efficient genetic redirection of human peripheral blood lymphocytes
Anthony Simmons1 & Kristina Jantz1
Abstract
Genetic redirection of lymphocytes that have been engineered to recognize antigens other than those originally programmed by their rearranged germlines is a potentially powerful immunotherapeutic tool. The rationale for the protocol described here is that many cancers and persistent or latent viruses have developed strikingly similar mechanisms of evading attack by host immunity that can often be overcome by redirection of host lymphocytes using chimeric T-cell receptor (chTCR) genes. However, for human peripheral blood lymphocytes (PBLs), this is generally regarded as a technically demanding procedure with unacceptably low efficiency using either contemporary transfection methods or retroviral transduction. One of the main difficulties with retroviruses is their reliance on rapidly dividing cells for integration of their genomes carrying the desired chTCR. Here we describe a highly efficient protocol that uses a lentivirus/vesicular stomatitis virus pseudotyped virus to engineer CD3/CD28-stimulated human peripheral blood cells (i.e., primarily T cells), with near 100% efficiency.
- Division of Virology, Children's Hospital, 301 University Boulevard, Galveston, Texas 77555-0372, USA.
Correspondence to: Anthony Simmons1 e-mail: ansimmon@utmb.edu
nature-products
A-Z product listing
- 10
buffer(Roche) - 24-well non-tissue-culture-treated plates(Falcon)
- 2 BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer(Sigma Aldrich)
- 5414R bench-top centrifuge(Eppendorf)
- 8-channel pipette 30–300
l(Eppendorf) - 96-well automated cell harvester(Brandel)
