I want to overexpress a mouse protein into mouse peritoneal macrophage, so which method will work best viral transduction or plamid transfection and if anybody has protocol for the same please let me know.
I believe mouse macrophages are difficulty to transfect (similar to BMDMs) and I would recommend transduction to express your protein of interest. Usually transduction can be done 8- 24hrs followed by antibiotic selection get a stable cell line with the over- expressed protein. The virus is usually made in HEK293T cells but it depends on the type of viral system you have (e.g. vectors) working in the lab.
If the protein is easy to express and purify in 293 or other cells, you may construct TAT-POI fusion plasmid and express the protein, and then purify TAT fusion protein. When you want to increase intracellular target protein, just add the fusion protein into culture medium. The fusion protein will goes into cell.
FYI....virus transduction.....lower level and long time expression;
plasmid transfection......higher level and short time expression.
If regular transfection does not work well you may try reverse transfection.
For detailed protocol you may check Current protocol in molecular biology...et al.
Dear Komal,
You have two simple ways to overexpress the protein:
1) The Lac Operon to produce T7 RNAP that will produce large amount of the protein
2) cloning viral promoters such CMV promoter ( strong promoter ) to overexpress the protein.
My best regards
Muhammed
CMV promoter is not good for long term stable expression in macrophage, but very good for transient expression.
For peritoneal macrophage, it is not as easy as BMDM or macrophage cell line to overexpress a protein since peritoneal mac does not proliferate any more in culture (?). So I think electroperation of a CMV promoter-containing plasmid is better than virus transduction (a little long time expression), but you can try if you like.
@ Fraser Soares: Can you please elaborate on: how to get stable cell line from primary mouse macrophages, as literature says that they don't proliferate in culture.
I guess it depends on the purpose of the experiment. If you are looking for purification of a protein then viral transduction is good as you can get many cells to express the O/E protein. We usually transduce BMDMs to do some short term experiments (e.g. one week experiments) for more functional/cell biology experiments. Selecting the O/E protein in your cells using antibiotics helps generate a stable cell line (as the virus usually infects a higher number of cells vs transfection). In addition we use oncogenic transformation (Myc, Ras or SV40) via transduction to generate a stable immortalized cell line that allows easier working conditions with the BMDMs. Again, these cells are immortalized cells so they might behave different from primary cells. Nevertheless, the immortalized cells are a great tool to investigate certain biological processes.
Hope all that helps.
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