Hello, i am currently trying to overexpress the CSF-1R receptor in the THP-1 cells in order to create a cell line to be used for the testing of CSF-1R inhibitors. In order to achieve this i am unsure what form of vector i should use, I require a GFP to be encoded by the vector along with the sequence of the CSF-1R.
Hello Rylan,
We have made a cell line expressing mCherry or a fusion protein of mCherry with lentivirus vector (CSII-CMV-Zeo modified)-infection. We couldn't use limited dilution or the other selection, so we still have a mixture of the non-fluorescent and fluorescent cells (aprox 80% positive), you may need to keep them in the selection antibiotics for long time because THP-1 might not proliferate from the single cell. Thus, without fluorescence, it is hard to monitor the quality of the overexpressing cells. Once I almost lost the overexpressing cells without the antibiotics. However, you may be worried if the fusion protein works properly, right? So the best way would be a fusion of CSF1R-P2A-GFP (self cleavable) or CSF1R-P2A-GFP-puromycin.
Low efficiency of the transfection is also the problem. So far TransIT-Jurkat cell transfection kit works well. The efficiency of Neon electroporation was much better, however, I don't know why, it did not survive longer after the antibiotic selection.
Hopefully many knowledge will be gathered here.
Good Luck for your experiments,
Sei
You can use a retroviral vector such as MIGR1 with IRES-GFP and clone in the sequence of CSF1R (many companies also will do this for you if you do not want to do the cloning). Transfection of 293T with appropriate amphotropic packaging will give you virus to infect THP-1 cells. This transduction approach would provide stable expression of CSF1R with GFP. If you sort (FACS) the GFP+ cells you can get a pure CSF1R overexpressing population to test your inhibitor. After you sort the GFP you can also viably freeze the THP1 so you do not have to repeat the transfection/transduction more times.
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