I need to establish a cell line that will express a bicistronic reporter. I have the reporter expression plasmid in hand. I have transiently transfected this plasmid into mouse macrophage RAW_264.7 cell line. I treated with LPS and got dominant expression of one of the 2 reporters and I treated with IL4 and got dominant expression of the other reporter so that is well.
To be of much greater use to me, I would like to establish a stable reporter cell line in RAW or in any mouse macrophage line. My reporter plasmid allows for puromycin selection.
What I am really looking for here is actual protocol for doing this.
Ideally you would introduce your gene of interest into a retroviral transfer vector like pQCXIP or pBABE. These can act as retroviral genomes. If you transfect such a plasmid along with MLV-Gagpol and VSV-G expression construct into 293 T cells, you will be able to obtain retrovirus stocks that contain the gene of interest, and upon transduction of Raw264.7 cells with the viral stocks, you will get stable cell lines that express your gene of interest and the cells can be selected with puromycin. This should be an extremely reliable approach
On the other hand, with your transient transfection, there is a low chance of integration of your plasmid into the cellular genome. If you select the cells with puromycin for 2 weeks, you will be able to select for cells that maintain the introduced plasmid stably. You will also have to maintain them under puromycin selection during subculture.
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