Background Flashcards
Describe packaging cell lines compared to transient transfection.
Packaging cell lines - A cell line that is stable and releases the desired product into the supernatant which is then harvested. Limited by the long creation time and the inability to produce proteins that are cytotoxic.
Transient transfection - introduced plasmids are not integrated into the genome. Advantageous because it is quicker and toxic transgenes such as HIV-1 proteins or V-SVG glycoprotein.
What processes are used for large-scale production?
Transient transfection of suspension cultures grown on a wave. Cultures are serum-free and made to GMP.
Why use his6?
The His6 tag is the most widely used meaning antibodies are available. It has also therefore been rigorously evaluated for purification.
Trade off between strong binding and elution.
How many bonds made per bead?
Difficult to predict but not that important for our study.
Current gold standard for purification
AEX, TFF
Is the His tag immunogenic?
Report that tag altered immune response of a vaccine.
What has the pseudo typed vector previously been used for?
CFTR induction in mice for upto 2 years.
What clinical trials are employing lentivirus?
Wet macular degeneration - Oxford Biomedica using EIAV.
Others targeting the thalsemias