I would like to perform over-expression experiments of tagged proteins using pcDNA3.1 as a vector. Keyword searches unvariably give me information on the use of vectors to induce pluripotency, but that is not what I am looking for. Any information will be appreciated. Thank you!
Hi,
The best for iPS transfection is nucleofection using Amaxa dedicated procedure. You resuspend the cells using Accutase or Tryple after ROCK inhibitor treatment, you follow the protocole and you seed iPSC at high density with ROCK inh to ensure cell survival. Be careful that overexpression in pluripotent cells often leads to high cell mortality independently of the gene of interest.
I hope this will help.
All the best in your research
Philippe
Hi,
The best for iPS transfection is nucleofection using Amaxa dedicated procedure. You resuspend the cells using Accutase or Tryple after ROCK inhibitor treatment, you follow the protocole and you seed iPSC at high density with ROCK inh to ensure cell survival. Be careful that overexpression in pluripotent cells often leads to high cell mortality independently of the gene of interest.
I hope this will help.
All the best in your research
Philippe
Dear colleague, I know that transfection plasmidic vector or infection by retroviral construction can be made during the time course of differentiation from the IPSC to the differentiated cells of interest but to transfect IPSC in culture seems to be difficult because they grow as colonies and they have to be dissociated to be transfected. Then it could be difficult to put it again in culture because they diffferentiate easily! It depends on if you want the IPSC to stay in un undifferentiated state or not.
Hi Josephine. I haven't tried to transfect hiPSCs before, but according to my previous experiences with human somatic and MSCs it is harder to transfect a stem cell than a somatic cell. So transfection of iPSCs can be even harder than the MSC.
I think you can try to transfect your genes before reporgramming, if it is available for you. You can use a dox-inducible system for your genes, for example. And activate transgenes after iPSCs were generated. But I am not sure if this may work. And I dont know it has ever done, but if not and if it works it could be a new method, worth publishing in a high rank journal.
Good luck with your research.
Well for transfecting iPSCs, you can use the amaxa nucleofection kit. They give instructions as to which program and which solution you can use for efficient transfection. You can check the lonza website for details. Good luck!!
It is possible. Culture conditions have a significant effect on efficiency. I've had best results about 24 hours post split when colonies are small and using a media such as NutriStem that allows colonies to remain flatter. You can also play around with using ROCK inhibitor to increase survival.
We have good results with the Lonza/Amaxa system as well for human iPSCs. For each line you will have to test out the conditions buffer/programs. Use Rock inhibitor, be gentle, work quickly. Attachment after nucleofection can be an issue. We had best results plating the cells on mouse embryonic feeders. Low oxygen conditions, if available, can also help.
Thank you all for your answers. I wonder if anyone has had acceptable results using Lipofectamine 2000 as a transfection agent.
As Kerry Mahon suggested I'll try transfecting when colonies are small in the presence of ROCK inhibitor, then go on to other methods if that doesn't work.
You can also consider a magnetic nanoparticles based transfection technology called Magnetofection, this review summarize the success of this method in many model including those with colony formation
http://www.ncbi.nlm.nih.gov/pubmed/21893135
Good luck
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