Hello
I'm trying to transduce some of AML cell lines with luciferase. The method I'm using is retrovirus transfection of 293T cells followed by transduction of the cells with help of Retronectin. The problem is that I get low transduction efficiency (less than 5%), and with one cell line (P31/Fuj) I get almost 0% transduction although I did transduction twice and got same results.
Any suggestion would be helpful.
Thanks
the description of your experiment is not very clear.
Do you mean that you are using a retroviral vector produced by transfection of 293T cells? Do you have a control for 293T cell transfection efficiency? If you are not using a packaging cell line, are you also cotransfecting a packaging plasmid and an envelope plasmid? Which envelope are you using? What is the approximate titer of your vector on a highly permissive cell line like NIH 3T3?
Hi Andrew and thanks for your reply.
Yes I am transfecting the 293T cells with a retrovirus vector encoding for Luciferase, and I use the non transduced cell line as a negative control. The marker I use for luciferase is the GFP to determine the efficiency by flow cytometry. In addition for the luciferase plasmid, I am also using pkat-gag-pol plasmid for packaging.
I hope the info is now better and sorry for the incomplete description.
You haven't specified your transfection efficiency, and that could have a large impact on the resultant production of retrovirus. It is preferable to have high virus titers in advance of transduction, and it could be that you just have low amounts of virus. Try using electroporation instead of transfection, which should be just as efficient for suspension AML cell lines (see https://altogen.com/product/k562-electroporation-kit-aml-leukemia-cells-ccl243/). Make sure to have high efficiency (experiment with conditions) and you should end up with better results.
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