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Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus‐based lentiviral vectors
Author(s) -
Kuate Seraphin,
Wagner Ralf,
Überla Klaus
Publication year - 2002
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.290
Subject(s) - viral vector , vesicular stomatitis virus , transfection , plasmid , simian immunodeficiency virus , biology , cell culture , virology , vector (molecular biology) , transduction (biophysics) , virus , helper virus , gene , expression vector , viral replication , genetics , recombinant dna , biochemistry
Background Lentiviral vectors allow gene transfer into non‐dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing. Methods To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon‐optimized gag‐pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV‐G) under the control of an ponasterone‐inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized. Results The RT activity and vector titers of cell clones stably transfected with the inducible gag‐pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone‐inducible VSV‐G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone‐induced producer clones vector titers of more than 1×10 5 transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production. Conclusions The packaging cells described should be suitable for most preclinical applications of SIV‐based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd.