I have established stable cell lines in the past by plasmid overexpression and also by crisper technology. Im new to lentivirus stable cell line generation an would appreciate any advice.
This is my understanding:
1. transfection of 293T cells with lentiviral vector and packaging plasmids
2. After 48hrs or 73 hrs, collect the supernatant and spin at 3000g, RT, 15min
3. Filter the supernatant through a 0.45um filter to remove cells and store at -80deg Celsius
4. grow target cells in 6 well plates. At 60% confluency, change o medium containing polybrene
5. Add 500ul or 1ml (depending on the virus titer) virus supernatant per well
6. After 24hrs change to fresh medium and incubate for another 24hrs.
7. 48 hrs after transfection change to medium containing antibiotic
8. 72 hours post transduction, the viral genome will be integrated into the host cell genome. Look at the cells for reporter expression (GFP).
My question is:
1. are only 293 T tells used to package the lentivirus vector with packaging plasmids or can we use our target cells?
2. the supernatant collected in step 3 can be used for transduction of any cell line?