I was wondering whether it is possible to transfect lentiviral vector like Tet-pLKO-shRNA vector (we are using Tet-pLKO-Puro vector) using Lipofectamine or Nucleofection. We aim to generate iPSC line using this vector for controlled shRNA expression, and would like to avoid any viral contamination. Any help or suggestions are appreciated. Thank you.
I have already transfected pLKO plasmids by nucleofection and also with jetPEI. If I were you I´d used lipofectamine rather than nucleofection for this type of cells. I think they have special lipofectamine to transfect this kind of cells.
Nucleofection is quite harmful. I´ve have problems transfecting lymphoma cell lines with this technique. You could try because the efficiency is very good, if you manage to transfect the cells without killing them. Many cells die during the electroporation process.
Hi Harshal and Ester,
Electroporation works very well for iPSC. The cell death caused by your "Nucleofection" is hard to avoid because cuvettes are used. If you use our new electroporation system, you won't experience such problems. All cuvettes based electroporation cause a lot of cell death due to physics reason explain in our webpage: http://www.celetrix.com/article_list_236.html
BTW, "nuclear transfection" is a coined wrong concept. All electroporation technologies can only move DNA across the cell membrane. It's impossible to deliver DNA directly into the nucleus due to virtual no electric field inside a living cell during electroporation. Explained here: http://www.celetrix.com/article_list_235.html
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