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Homepage of the Lentiviral Gene Ontology Vectors |
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{Why is Polybrene added in a transduction?}
Polybrene (also called Hexadimethrine bromide) is a cationic agent that is thought to shield the negative surface charges of virus an cell,
thus increasing transduction rates by decreasing repulsion. Depending on the cell type it may have a huge effect
and can increase the titer up to 10-fold. The standard concentration is 8 µg/ml in the cell culture medium during transduction
(stock solution: 8 mg/ml in water or PBS). Polybrene is toxic to the cells in higher concentrations and some delicate primary cells may not
like it at all. Lower concentrations of about 4 µg/mL may already show the maximal effect and if necessary the optimal concentration can be determined experimentally.
Also Protamine sulfate and other cations have been shown effective. Read more: PubMed-link.
{Why is Chloroquine added during the transfection?}
Chloroquine shall decrease the intracellular degradation of plasmid DNA in the lysosomes,
thus increasing the amount of plasmid DNA that reaches the nucleus and gets expressed. The standard
concentration is 25 µM in the cell culture medium during transfection (stock solution: 25 mM in PBS).
But I also had good titers when I forgot to add Chloroquine, I haven't ever measured the effect.
The literature says something like "great effect on some cells, no effect on other cells".
Read more: PubMed-link.
{Where do I get the packaging plasmids from?} |