In one of my project, I want to transfect 9 kb plasmid construct into a Myeloid progenitor cell line. I am facing much difficulty to get good transfection efficiency as well as good viability. Any input in this regard is greatly appreciated?
Hi Sundeep,
I agree with comments above difficult to transfect myeloid progenitor especially if DNA is large. Nonetheless, I can suggest DreamFect, it has been successfully used in THP1, jurkat, k562 http://www.ozbiosciences.com/transfection-dna/15-dreamfect-transfection-reagent.html. Good Luck
Did you try electroporation or lentiviral transduction? In our lab, although wiht low efficiency, lentiviral transduction is working for lymphoblastoid cell lines.
I already tried with couple of transaction methods such as Neon, Amaxa, Lipofectamine into these suspension cells. However, there is no improvement of transaction efficiency in these cells.
My experience is that lipid based transfection reagents do not work well in non-adherent cells. I did not try electroporation but, as I told you, we are using lentiviral transduction. In this regard you will have to clone your construct in a lentiviral vector (we are using pUltra). We obtained good transfection efficiencies with the empty vector and is less efficient once the construct is cloned. I hope that it helped, good luck.
a construct of 9 kb is quite large, and you may encounter problems because of the lentiviral packaging size constraints. If there is a way to (temporarily) increase the presence of adhesion molecules at the cell surface, e.g. by culturing the cells in the presence of ECM molecules, non-viral transfection may be enhanced.
Hi Sundeep,
I agree with comments above difficult to transfect myeloid progenitor especially if DNA is large. Nonetheless, I can suggest DreamFect, it has been successfully used in THP1, jurkat, k562 http://www.ozbiosciences.com/transfection-dna/15-dreamfect-transfection-reagent.html. Good Luck
you can try lipofectamine 3000 instead of 2000 its have great efficiency
For suspension cell lines (like RAW 264.7) electroporation will help you achieve higher transfection efficiencies (see https://altogen.com/product/raw-264-7-electroporation-kit-lymphoma-cells-tib-71/). This is especially the case if your cells are proliferating slowly. A 9 kbp plasmid shouldn't be too difficult to transfect, but it may lead to cell death from the excess penetration of the cell membrane.
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