I am reprogramming human dermal fibroblasts to iPSCs using Yamanaka factors with Nucleofection method. All obtained iPSC clones shows integration of one or a few plasmids after passage 6 of the iPSCs. The amount of fibroblasts and plasmids used were optimised as per the established protocol. Kindly suggest any possible troubleshooting steps for optimising the experiment.
Thanks in advance!
Dear Kalaivani Manibarathi
The Yamanaka four-factor (Sox2, Oct4, cMyc, Klf4) only reprogramming method may be show differences in efficiency and quality depending on the cell line being reprogrammed.
It would be more appropriate to use a method that is known to be less prone to integration (EBNA vector, Sendi virus, mRNA).
The addition of compounds or genes that serve as auxiliary factors to increase the initialization efficiency may also be effective.
Best,
Narumi
The plasmid vector would not be persistent enough to cause successful reprogramming. You need sustained expression for some weeks, in order to achieve reprogramming. If you transfect a regular plasmid, only clones with integrations are able to achieve that.
As Narumi pointed out, there are non-integrating methods available, but they're not based on plasmids.
While I agree with Narumi Uno's comment that the use of non-integrating methods is a good way to restart the experiment, it is might be possible to utilize your existing lines since the integrated plasmids might be silenced after several rounds of expansion. If you would like test whether some of your existing IPSC clones undergo silencing, just culture them for >10 passages and screen for expression of the introduced vectors. This is the fastest and easiest step forward. Otherwise, you restart and optimize the non-integrating system as mentioned above.
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