Hello,
I have been trying to transfer a few big size plasmids (~12kb) into primary pancreatic ductal cells which are epithelial cells. With small plasmids I had a very good transfection rate (~80%), but when tried with big vectors the transfection rate is less than 1% . So far I had tried lipofection and electroporation but none of them has worked. Does anyone have experience with big plasmids electroporation in epithelial cells? I highly appreciate any suggestion.
Your plasmid is pretty big, so I would recommend using a complex condenser in tandem with a primary cell transfection reagent (e.g. https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/) in order to boost overall results. Electroporation with such large plasmids can be pretty damaging, and lentiviral transduction can be complicated, but if you compact your plasmids (with a complex condenser) and use tailored reagents for primary cells you should be able to get better results.
That's a pretty remarkable difference... are you sure there isn't something wrong with the preps of your ~12kb plasmids? Or that it isn't a batch to batch effect of the primary cells (i.e. have you seen the 80%/1% discrepancy in aliquots of the same batch of cells transfected in parallel at the same time?). Leaving those aside, a lentiviral approach is probably the way to go.
I think, the best way is always manually transfection procedures like heat-shock.
I would try a chemical transfection instead of electroporation since you are working with primary cells. I would recommend to try one of the TransIT reagents of MirusBio reagents. I have to work with big size plasmids (~14kb) too and I have much better transfection rates since I'm working with these reagents. I also have tried nucleofector and other reagents such as lipofectamine. But I have a lot of cell death with electroporation and insufficient transfection rate with lipofectamine. My experience shows that this novel non-liposomal, polymeric delivery works the best for our goal. Maybe you can order some samples of the company to try some of the reagents to find out which of this TransIT reagent would also fit to your goal.
Thank you Aydin, I have never heard of using heat shock for mammalian cells. Can you please explain what you mean. Thanks.
Nicholas, thank you. I am quite sure it is not about the DNA prep. I had a high transfection efficiency using the same vectors in HEK293 cells. But I agree that probably a viral system is the way to go here.
Jaison, thank you. I tried Polyjet at the beginning, but unfortunately did not get a good transfection. But I will check MirusBio reagents. If it works it would save a lot of time and money for us.
Your plasmid is pretty big, so I would recommend using a complex condenser in tandem with a primary cell transfection reagent (e.g. https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/) in order to boost overall results. Electroporation with such large plasmids can be pretty damaging, and lentiviral transduction can be complicated, but if you compact your plasmids (with a complex condenser) and use tailored reagents for primary cells you should be able to get better results.
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