TALEN efficiency: electroporation versus "normal transfection"

[christiaa...@gmail.com]

Dear all,

I was wondering if someone has experience with the use of TALENs in induced pluripotent stem cells (IPS-cells) and could help me out here.
I used a TALEN pair shown to be efficient in HEK-cells (efficiency 25 %), however in the IPS celline efficiency is only 3%.
The efficiency in the IPS cells was tested 2 days after transfection (cotransfection with GFP for FACS of the transfected cells), by cloning the PCR product flanking the target side in a TOPO vector. Expression of the TALEN shouldn't be a problem: the vector I used is the goldyTALEN vector containing a CAG promoter.


One of my ideas was to perform electroporation in the IPS cells (instead of transfection with FugeneHD). As far as I know, the amount of DNA which can be used with electroporation is higher, which might lead to a higher dose of TALEN eventually. With my transfection, I used 7 ug of each TALEN in ~ 8-9 million cells, which is reaching the maximum. Most papers where TALENs were used in IPS cells perform electroporation, indicating it might be a better strategy. Anyone has some ideas about this issue?

Thanks.

Christiaan

[TALENAIR...]

Dear Christian,

Did you transfect your iPS cells with TALENs? Did you use Fugene or Micrporation for transfection? How were the results of transfection? How much DNA did you put in of each LEFT and RIGHT TALEN on iPS Cells.

Please let us know about the outcome.

Thanks a lot for your time.

TALENair

[aami...@gmail.com]

Hi Christian,

I also failed using FugeneHD, but was able to get stable expression using AMAXA Nucleofacter electroporation on iPSC. I used 0.8 ug of each TALENs in one well of a 24-well plate (1 million cells).

Good luck,

A

[christiaa...@gmail.com]

Thanks for your responses.
My transfection efficiencies with FugeneHD were allright (20 %), however I think the problem was the fact that I sorted by using a third plasmid, CAG-GFP (only selecting green cells). When I cloned my TALEN constructs in a IRES-GFP and IRES-dsRED, selected for double-positive cells, my efficiency was 30 % again.

Best, Christiaan

[TALENAIR...]

Dear Christian,

Did you transfect your iPS cells with TALENs with Micrporation for transfection? How were the results of transfection? How much DNA did you put in of each LEFT and RIGHT TALEN on iPS Cells.

Please let us know about the outcome.

Thanks a lot for your time.

[hyasuda...@gmail.com]

Hi, Christian;

Please try to use NEPA21 electroporator. This is new innovation from Japanese company. The efficiency is quite well (more than 60%). In this case you do not need to use selection marker (GFP or RFP).

Hideyo