Stable Retroviral Transduction
HEK2932 - making lentivirus
1. (Day 1) Plate 2.5 x 10^5 of 293T cells on a 10cm dish and incubate at 37C overnight.
2. (Day 2)
- In a labeled ependorf tube (vial 1), mix the following DNA with 500ul Opti- MEM
a. 5 ug of pLenti-shRNA construct or 5 ug of pLenti-ORF expression construct
b. 6 ug of packaging plasmids
- In a separate tube (vial 2), mix 44ul of MegaTran transfection reagent with 500ul Opti- MEM
- Transfer the DNA solution from vial 1 into vial 2 containing MegaTran. Vortex it and incubate 15-30 min at room temperature
- Add the mixture of DNA and MegaTran directly to the 10cm dish of 293T cells
3. (Day 3) After 12-18 hrs incubation, change the culture medium
4. (Day 4) Harvest the first batch of viral supernatant from the culture and store it at 4C. Add fresh culture medium to the cell culture.
5. (Day 5) - Harvest the second batch of viral supernatant then combine it with the first batch.
- Spin 3000rpm/min and filter through a 0.45 micron filter to remove cellular debris.
- The viral titer at this step is usually 10^8 - 10^7 TU/ml**. The viral supernatant is now ready for the majoruty of transduction applications. If necessary, further concentration can be applied. **Large ORF inserts will decrease the viral titer.
832/13 INS - 1 using lenti virus
1. Plate the target cells at a concentration that will produce approximately 50% confluency in 24 hrs
2. Add entire amount of viral stock ( or, if virus has been titered, the chosen cfu/mL of virus) and 4ug/ml polybrene in growth medium directly onto target cells. incubate at 37C in 5% CO2.
3. At 24 hours post-infection, replace the medium with fresh growth medium containing 0.5-1 ug/ml puromycin (or the optimal concentration as determined for your conditions). Passage as need, and maintain the selection pressure for 1-2 weeks. Most uninfected cells should be killed by the puromycin within 1 week.
4. Select clonal populations of cells by transferring a well-isolated single clump of cells (the clonal ancestor and cells divided from it) into a well of a 24-well plate; repeat to select 5-10 clonal populations. Continue growing the cells in selection medium for 1-2 additional passages. At this time each well contains a clonal population of stably transfected cells, which can be maintained in normal growth medium without the selection pressure of puromycin. These populations can be used for experiments or stored in liquid nitrogen tank on growth medium with 10% DMSO and 20% FBS for future use.