kat: a high-efficiency retroviral transduction system for primary human T lymphocytes
- PMID: 8274751
kat: a high-efficiency retroviral transduction system for primary human T lymphocytes
Abstract
We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to be free of detectable replication competent retrovirus by an extended provirus mobilization assay, demonstrating that this system is as safe as currently available stable packaging lines. The kat virus production system should be of general use for the rapid production of high titer viral supernatants, as well as for high-efficiency transduction hematopoietic cell types refractory to retroviral transduction.
Similar articles
-
Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols.J Gene Med. 1999 May-Jun;1(3):195-209. doi: 10.1002/(SICI)1521-2254(199905/06)1:3<195::AID-JGM31>3.0.CO;2-#. J Gene Med. 1999. PMID: 10738568
-
Generation of retroviral vector for clinical studies using transient transfection.Hum Gene Ther. 1999 Jan 1;10(1):123-32. doi: 10.1089/10430349950019255. Hum Gene Ther. 1999. PMID: 10022537
-
Improvement of retroviral packaging cell lines by introducing the polyomavirus early region.Hum Gene Ther. 1998 Jan 20;9(2):161-72. doi: 10.1089/hum.1998.9.2-161. Hum Gene Ther. 1998. PMID: 9472776
-
Production of high-titer retroviral vectors and detection of replication-competent retroviruses.Mol Cells. 1998 Feb 28;8(1):36-42. Mol Cells. 1998. PMID: 9571629
-
Transduction efficiency of pantropic retroviral vectors is controlled by the envelope plasmid to vector plasmid ratio.Biotechnol Prog. 2005 Jan-Feb;21(1):274-82. doi: 10.1021/bp049865x. Biotechnol Prog. 2005. PMID: 15903266 Free PMC article.
Cited by
-
CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy.Clin Exp Immunol. 2014 Feb;175(2):258-67. doi: 10.1111/cei.12216. Clin Exp Immunol. 2014. PMID: 24116999 Free PMC article.
-
Pseudotype-based neutralization assays for influenza: a systematic analysis.Front Immunol. 2015 Apr 29;6:161. doi: 10.3389/fimmu.2015.00161. eCollection 2015. Front Immunol. 2015. PMID: 25972865 Free PMC article. Review.
-
Cyclin-dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis.Cell Prolif. 2007 Oct;40(5):721-40. doi: 10.1111/j.1365-2184.2007.00463.x. Cell Prolif. 2007. PMID: 17877612 Free PMC article.
-
The adaptor protein ARH escorts megalin to and through endosomes.Mol Biol Cell. 2003 Dec;14(12):4984-96. doi: 10.1091/mbc.e03-06-0385. Epub 2003 Oct 3. Mol Biol Cell. 2003. PMID: 14528014 Free PMC article.
-
Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J Virol. 1998 Dec;72(12):9873-80. doi: 10.1128/JVI.72.12.9873-9880.1998. J Virol. 1998. PMID: 9811723 Free PMC article.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
