Hi,

I have successfully used the PiggyBac cumate inducible vector in U2OS cells using Extreme Gene 9 from Roche, and I have used the constitutive active PiggyBac EF1-MCS-IRES-NEO vector successfully in U2OS, hTert-RPE, and NRK cells.

The protocol I used was pretty much the same for both:

1) plate cells in 6 well dish so they will be at ≈50% confluency at 24 hrs (total volume of mead in well is 2mL, medium is DMEM with 10% FBS) 

2) Next day transfect with Extreme gene 9, in an eppendorf tube combine:

100µL OptiMEM

3µL  Extreeme gene 9

0.2µg PiggyBac Transposase

0.5 µg Transposon vector construct with your gene of interest

Incubate for 15min RT then add to wells drop wise and put back in the incubator for 48hrs or until 100% confluent

3) Transfer 6 well to 10cm, let recover for 24 hrs

4) next day add selection media containing Puro (U2OS only take about 1.5µg/mL to select you would need to do a kill curve for the HEK cells)

5) wait 6-7 days for selection, change media half way through (there should be a lot of cell death, my transfection was fairly low, but if you select and grow up it should be fine)

6) remove selection media and add back regular media and let cells grow back up

When the cells were grown I froze down a portion, used a small amount for serial dilution to make clonal cells, and put a small amount on 2 cover slips in a 24 well dish and induced one while not inducing the other (also good to have a non-transfected control) then let it go for a few days to see if I could see induction (note: I took out the IRES GFP because it would get in the was of my FRET construct that I was inducing, so I couldn't see anything until I induced). The cumate system is actually kind of slow, so unless a large number of genes made it in, then it can take a while (the system biosciences   website shows nice bright cells, but if you notice that is after 11 days).

I have also tried a few different ratios, but didn't feel it made all that much of a difference. Although I have read that changing just the transposon vector amount is more likely to affect the number of inserts than adjusting the transposase.

One thing that is probably unlikely, but you may also want to check if you grew up the transpoase is whether or not your transposease vector is propagatable in bacteria. This will depend on when you purchased the transposase because the company which systembiosciences licenses the transposase from, made the transposase non-propagatable about a year ago (February 2014). I ran into this problem because unfortunately I purchased it right when the licenses were changing, so I got the documents for the old propagatable transposase with the new non-propagatable transposase vector. To top it off I was still able to get some colonies (very few, about 5 when I got hundreds with the transposon vector) when I transformed it (the company said it was probably due to re-combination events). So I tried for quite a while with a what I thought was the transposase, but wasn't. Once I finally used the actual transposase though it was fine...

Also, a note on transfection reagents, is that I have always had better luck with Extreme Gene 9 or Fugene 6 (I feel they are fairly interchangeable in my hands) than lipofectamine.

Good luck!

Kari thanks a lot! We ordered now the SBI transfection reagent, we thought it should work if we do it just like they say in their protocols.. If not than I'll go back to your advise.

HI Vanessa,

Your welcome :) Hopefully you get it working one way or the other.

Also If you continue to have trouble, I would recommend contacting SBI and specifically ask to set up a phone meeting with Travis Antes at SBI. He is currently their Director of Product Management, and of all the people I spoke with for technical problems he was the most helpful.

Hi Chris,

i was using the same Vector and it turned out, that there was a mutation in my Vector, due to the competent cells I used. SBI suggests special cells which dont recombinate, especially useful when using large inserts. So maybe you should have a look at that first :)

For my experiment in the end I gave up on the PiggyBac but I'm going to get it started again soon. Maybe then you can give me some advice :)

Hello Vanessa Dietinger ,

Did you manage somehow your PB transformation to work?

I'm asking this because I started to use the PB cumate-switch plasmid. The transformation seems to be OK but I didn't get any GFP or GOI expression.

Thanks,

F

Hi Farkas,

unfortunately it has been a while since I finished this porject. If I remember correctly, I started with different cells back then which was working better, eventhough efficenty wasnt that high.

Have you checked if your vector is correct?

Best,

V.

"Have you checked if your vector is correct?" - I didn't sequence the whole vector, only my GOI. I also digested the vector+GOI and it showed the right sizes.

If you meant checking like this.

I also checked the sequence of the vector. This is the

PB-Cuo-MCS-IRES-GFP-EF1α-CymR-Puro all-in-one vector ( https://www.systembio.com/products/gene-expression-systems/piggybac-transposon/pb-cuo-mcs-ires-gfp-ef1-alpha-cymr-puro/). As it is not shown on the map, there is no terminator/polyA site after the GFP. Only after the second casette. I think there should be but I might be wrong.

If you meant checking like this.

Thanks for your time :)

May You kindly provide me with

PB-Cuo-MCS-IRES-GFP-EF1α-CymR-Puro sequence ?