Dear Nicole,

Upon transient transfection plasmids are generally lost within 72 hours, that could explain why your cells are dying at such high rate when you apply puromycin. Probably the 0,5 % of cells that survive your selection process are the ones that managed to stably integrate the plasmid into the genome.

Starting selection at 48 after transfection for additional 48-72 hours could solve the issue. Alternatively you can use Cas9 plasmids with a fluorescent reporter (such as GFP) to follow up transfection and check when the plasmid is lost. 

Another viable strategy is to co-transfect your cells with a GFP plasmid together with your Cas9 Puro plasmids. When GFP starts to be expressed (usually 24-48 hrs after transfection) you will reasonably know that your Puromycin resistance gene is being expressed too, so you can safely apply selection. If you wait too much after transfection plasmids will be diluted, and a lower percentage of cells will survive selection, specially if your cells divide fast.

Hope it helps

Good luck

Francesco

Dear Nicole,

Upon transient transfection plasmids are generally lost within 72 hours, that could explain why your cells are dying at such high rate when you apply puromycin. Probably the 0,5 % of cells that survive your selection process are the ones that managed to stably integrate the plasmid into the genome.

Starting selection at 48 after transfection for additional 48-72 hours could solve the issue. Alternatively you can use Cas9 plasmids with a fluorescent reporter (such as GFP) to follow up transfection and check when the plasmid is lost. 

Another viable strategy is to co-transfect your cells with a GFP plasmid together with your Cas9 Puro plasmids. When GFP starts to be expressed (usually 24-48 hrs after transfection) you will reasonably know that your Puromycin resistance gene is being expressed too, so you can safely apply selection. If you wait too much after transfection plasmids will be diluted, and a lower percentage of cells will survive selection, specially if your cells divide fast.

Hope it helps

Good luck

Francesco

Hello Nicole,

It seems you have used too high a concentration of selection antibiotic. Usually we choose antibiotic concentration that kills 100% untransfected cells in 7-10 days. That said, it is quite possible that transfection efficiency was not good and only a small percentage of your cells got transfected. I ll suggest you to re-do the kill curve and select antibiotic concentration as described above. Like Franscesco said, it would be better to have some kind of reporter gene cloned in your plasmid so you could optimize your transfection protocol. Alternatively, use a similar sized plasmid vector harboring a reporter gene and optimize your transfection protocol. Use the optimized protocol to transfect cells with the desired plasmid. It is not ideal but it will help.

Well, we use the Santa Cruz Nickase Protocol. It includes a GFP reporter gene plasmid. I checked the transfection rate before I added the selection media. I had many weakly fluorescent cells and only very few brightly fluorescent cells. I am not sure if the weakly fluorenscent cells have incorporated the plasmid or if it was only background fluorescence.

Talking of Santa Cruz... How was the transfection with the control plasmid?

How old were your transfected cells? My experience is in case of generating stable transfected cells using the TET ON 3G system that they should be pretty new thawn as otherwise you will never get a really good transfection indicated by only weak positive green stained cells.

Apart from this, I was going for a dose titration of my antibiotic to obtain a suitable concentration. And I think that I started with antibiotic treatment after 48 hours.

The transfection with the control plasmid was very low. In the meantime I got two colonies which I let grow now. Let's see is it harbors what I am looking for.

Dear Nicole,

I just made stably transfectants of ShSY5Y and got a good number of clones with very different properties regarding the transgene. I am very interested how the cells from your clones will be! Good luck and best wishes, Erik

Dear Erik,

Okay, I will keep you informed. I also started a new transfection round, used less puromyin (5 µg/ml) and hope for the best. How much plasmid did you use? I am using 1 µg with 2.5 µl Lipofectamine 2000 in OPTI-MEM. Did you use the special media the companies are suggesting? Thanks Nicole

Dear Nicole,

I plated 200.000 cells/well in a 6-well plate and transfected them with 1.5 µg DNA in 7.5 µl FuGene HD in 300 µl OptiMEM (One fifth of this preparation to 2 ml SHSY5Y cells = one well in DMEM/10%FCS and GlutaMax), left them over night and treated them with 250 µg/ml Hygromycin for 4 days before medium change. At the end of this week the first colonies showed up. I didn`t use any special media beside OptiMEM. This was my procedure for HT22 cells and another mouse neuronal cell line (where it worked, too). You may just give then cells more time, some may not be dead, but just lokk as if...

Best, Erik

Dear Erik,

I promised to give an update. In the end I got colonies which were GFP-positive and some of them had a knock down. So we thought that maybe a FACS sort would give us a knock out. Unfortunately, it led us to losing the knock down, strangely and unexplainably. Now, I am trying a new approach where I do the FACS sort shortly after the transfection in order to obtain more GFP-positive colonies. 

That the cells sometimes look as if they were dead I also observed. After some time they are suddenly a colony, only that I have the impression that somehow they are all false negative. 

Let's see what the new approach will bring.

Best Nicole

Dear Nicole,

thanks for the update! False surviving cells I also had sometimes, thats why I always check subclones functionally. Good luck with your modified procedure!

best wishes,

Erik

10 ug/ml puromycin is way too high for SY5Y cells. In my hands 2 ug/ml already killed mock transfected cells in 2 days.