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.

Transplanted CNS stem cells form functional synapses in vivo

“…Retroviral infection of precursor cells in vitro with replication-defective GaLV particles carrying the gene for GFP ( Fig. 1A, see also Materials and methods; Finer et al, 1994) ensured that only proliferating cells were labelled. Following in utero grafting of these genetically labelled, GFP-expressing, CNS precursors into the developing hippocampus ( Fig.…”

“…After 4 days in vitro the cells were exposed for 4 h to medium containing replication-defective gibbon ape leukaemia virus (GaLV)enveloped retroviral vectors. GaLV vectors were produced using the kat system (Finer et al, 1994). GaLV envelope and gagpol genes were inserted into pkat1 plasmids optimized to produce high levels of retroviral transcripts when transiently expressed in human 293 cells.…”

Cyclin‐dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis

“…We used the Kat retroviral vector (Finer et al 1994) to transduce genes into cells. This was originally supplied by Cell Genesys (Foster City, CA, USA) and was subsequently modified by introduction of an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) sequence to form rKat.IRES.EGFP (Gilham et al 2002).…”

The prodomain of interleukin 1α interacts with elements of the RNA processing apparatus and induces apoptosis in malignant cells

“…A 1100 bp fragment encoding the IL-1␣ propiece-EGFP fusion protein was ligated into pRT43.267, a retroviral vector designed to produce high titer preparations in transient transfections (10). A control retroviral vector contained only the coding sequence for the EGFP protein.…”

“…Because the efficiency and toxicity of conventional transfection reagents is very cell specific, simple plasmid-based transfection applied to 60 discrete cell types could introduce considerable artifact, especially as to toxicity. Therefore, the IL-␣-EGFP cassette and the EGFP control were placed within a retroviral expression vector (10). Using retrovirus pseudotyped with the VSV G envelope protein prepared at high viral titers (11,43), IL-1␣ was expressed in all members of the cell panel.…”