Stable Lentiviral Packaging Cell Line

Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors.

Stable lentiviral packaging cell line.

This uses cre recombinase mediated cassette exchange to insert a codon optimised hiv 1 gag pol expression construct in a continuously expressed locus in 293ft cells. Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein vsvg pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293t cells with multiple plasmids. This represents the first nontransient production protocol using a sin vector packaging cell line. We have successfully generated and characterized a stable packaging cell line for hiv 1 based vectors.

Lentivirus and other retroviral vectors are useful tools to engineer chimeric antigen receptor car t cells which are designed to target tumor cells in the growing field of gene therapy. Lentiviruses are used widely to generate stable expression mammalian cell lines. This study demonstrates that csin transduction of sodk1 stable packaging cells 11 results in a stable sin lentivirus vector producer line from which vector titers higher than 10 6 iu ml can be routinely generated. To allow safe production of vector a minimal packaging construct carrying only the coding sequences of the hiv 1 gag pol tat and rev genes was stably introduced into 293g cells under the control.

Overall the availability of high titer idlv lentivirus packaging cell line described here will significantly facilitate idlv based basic science research as well as preclinical and clinical applications. The icellis is a packed bed bioreactor for adherent or entrained cells with surface areas from 0 53 to 500 m 2. Lvv are typically produced in hek293 t cells in suspension bioreactors using serum free media or adherent cultures with serum. Based on the drop in ph post induction for the gprtg ef1α hγ c opt stable cell line in cell factories and studies that have shown transient lvv production to increase at lower ph values 29 31 the effect of maintaining a range of ph set points throughout the vector production phase was investigated.

They are used for gene down regulation by using shrna or for gene up regulation by using orf of the gene of interest. Reactors were inoculated at 15 000 cells cm 2 8 10 7 cells total and maintained at ph 7.