I found a very interesting protein(gene) and want to study its function. When I overexpress this protein in the cell by lentivirus, do i need care about the insert copy of the gene? Or just control the MOI carefully?
Also, I used the lentivirus to overepxress other protein(GFP selection marker), but I found the protein will lost during continus cell culture, what's the method to avoid that?
Very appreciate if you can give me suggestion.
Depends on the lentiviral system you are using. Once the cells are infected plasmids containing fully functional lentiviral replication machinery will replicate to around a certain copy number. Some systems have mutated origins for reduced copy number or are binary systems where packaged DNA lacks genes needed for origin function which are on a non-packaged helper plasmid.
Ideally on your plasmid you should only have one copy of your gene of interest.
Unless you have a cell sorter to allow easy GFP selection, you may want to use an antibiotic resistance marker. G418, puromycin and blasticidin are common ones. Check that your lentiviral system does not already have a resistance cassette for one of those.
You can manipulate the MOI to help ensure that cells only receive a single copy of the transgene after you perform a selection. I'd recommend using an antibiotic selection system rather than FACS, unless you want to expand your cell line from a single cell clone.
If you're not keeping your cells under selection or working with a single clone, you may see a dropout of your reporter and/or transgene.
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