Tet-on PA-1 cell line
Transfection and selection conditions:
Tet-on PA-1 cell line - LD208 - rtTA - G418
Seed: 80 000 cells/well - 3 wells (from 6-well plates) (Thursday 06-14-18)
Transfection: 24 h post-seeding (Friday 06-15-18)
- 3ul of Xtreme 9 + 97 ul OptiMEM (prewarmed) - Incubation 5 min at RT
- 1ug LD208 - rtTA - G418 plasmid + 100 ul Xtreme 9-OptiMEM mix - Incubation 20 min at RT
- Add 100 ul of plasmid+Xtreme 9+OptiMEM per well (drop by drop)
- Overnight incubation at 37ºC.
Change media the next day - Selecction with 200 ug/ml of G418 (Saturday 06-16-18)
Cell pass from 6-well plates to 100 cm dishes (Monday 06-18-18).
9-10 days after selection (06-25-18) split cells doing dilutions 1:50 and 1:1000. Freeze the rest of the pool.
1:1000 dilutions didn't survive selection.
1:50 dilutions did but had around 50 clones.
Cells grow very slow when they are alone.
Picked 46 clones (07-03-18)
Tet-on PA-1 cell line - LD215 - rtTA - BSD
Seed: 80 000 cells/well - 3 wells (from 6-well plates) (Thursday 06-14-18)
Transfection: 24 h post-seeding (Friday 06-15-18)
- 3ul of Xtreme 9 + 97 ul OptiMEM (prewarmed) - Incubation 5 min at RT
- 1ug LD215 - rtTA - BSD plasmid + 100 ul Xtreme 9-OptiMEM mix - Incubation 20 min at RT
- Add 100 ul of plasmid+Xtreme 9+OptiMEM per well (drop by drop)
- Overnight incubation at 37ºC.
Change media the next day - Selecction with 5 ug/ml of Blasticidine (Saturday 06-16-18)
Cell pass from 6-well plates to 100 cm dishes (Monday 06-18-18)
On Wednesday cells weren't dying... 5ug/ml BSD selection wasn't very effective. Split cells
9-10 days after selection (06-25-18) split cells doing dilutions 1:50 and 1:1000. Freeze the rest of the pool.
1:1000 dilutions didn't survive selection.
1:50 dilutions did but had around 50 clones.
Cells grow very slow when they are alone.
Picked 48 clones (07-03-18)
This are the procedures followed to transfect the cells. We tried two times.
What we learnt:
- PA-1 cells are not happy if they are very diluted or very confluent. Best dilutions to get colonies were between 1:20 and 1:100.
- Antibiotic selection occurs slowly (but still seems to be effective - Paolo thinks we can increase them a little bit, but there shouldn't be a big difference after the tests that we performed) Select the cells for at least 15 days (or until cells stop dying) before picking colonies.
Blasticidine: 5 ug/ml
G418: 200 ug/ml
- To be sure that the cells were selected, the second time, we let the selection occur for at least 15 days. Meanwhile, we splitted the cells at least 3 times with dilutions 1:2, 1:4 and 1:6.
- Best conditions for picking cells were:
5 minutes incubation at RT + neutralization with a mix of fresh media/conditioned media 1:1. Pick the cells and transfer them to 96-wells with 100 ul of a mix of fresh culture media/conditioned media 1:1.
Plasmids used:
- LD208/PM65= rtTA advance G418 selection (clontech)
- LD215/PM66=rtTA advance BSD selection (Lixin did this: pLD208 Neo gene was replaced by BSD gene from pLD205. Xho I for vector, XhoI-SalI for insert)
Next step:
Start from the beggining applying what we learnt.
Option: Using linearized plasmids for transfection (Marty thinks this could help, however Paolo thinks it doesn't matter, because we got cells/selected cells, the problem was that the colonies we picked didn't survive).
Date: 08-14-2018
Paolo kept the cells from the second transfection growing to pick colonies (in case he succed)
The cells are growing.
Last week I picked 96 rtTA BSD clones that are now growing pretty well and yesterday I picked 96 rtTA neo for which I can see cells attached to the bottom of the plate today (as you know though I need to wait at least a couple of days to see if they are really growing because usually they die the within 2 days from picking).
So I think that in max a couple of week I will have the result of the testing for both cell line.