Stable Cell Line Generation Using Lentivirus

The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1 2 weeks to get stable expression for most mammalian cell lines.

Stable cell line generation using lentivirus. Express the hiv gag pol rev and tat genes all from a single packaging plasmid such as pspax2. 2nd generation packaging systems. Generation of stable cell lines using lentivirus. The cell line is created with stable transfection of bicistronic expression cassettes with re initiation of the translation mechanism.

The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1 2 weeks to get stable expression for most mammalian cell lines. They are used for gene down regulation by using shrna or for gene up regulation by using orf of the gene of interest. Furthermore a third generation lentivirus vector stable packaging cell line deficient for the hiv 1 tat gene was successfully used to generate high titer csin vector stocks following transduction with the ptk136 csin vector kafri unpublished data. Unlike the short term protein expression observed using transient transfection approaches generating cell lines using lentiviral vectors enables long term protein expression studies.

Lentiviral vectors offer stable integration of your gene of inte. The authors describe a new lentiviral vector packaging cell line lentipro26 using a mutated less active viral protease to overcome potential cytotoxic limitations. The advantage of using the 3rd generation lentivirus are that are very safe and they are replication incompetent. This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector.

Shrna allows for stable knockdown of genes while sirna allows for transient knockdown. They are used for both gene down regulation by using shrna or for gene up regulation by using orf of gene of interest. By contrast lentiviral vector supply based on stable producer cell lines involves added complexity at the start of the drug development project through the need to perform a cell line development campaign and establish gmp mammalian cell banks but has benefits later in terms of a simpler supply chain and a simpler upstream process with fewer. Lentiviruses are used widely to generate stable expression mammalian cell lines.

Briefly 2nd generation lentiviral systems use more hiv proteins on fewer plasmids in order to produce functional lentiviral particles than 3rd generation systems.