Anyone has idea about the unexpected non target gene expression (or extremely low expression) by lentiviral transduction, though GFP could be expressed by the same lentivector (same vector,e.g. promotor, kozak, etc)? Is GFP too easy a protein for production by any vector?
Another point to consider: if your pet protein is detrimental to the cells, they can easily methylate the CMV promoter, or employ a number of more exotic survival strategies. Did you check if the RNA is expressed?
A bicistronic pet-IRES-GFP or a self-cleaving polypeptide pet-P2A-GFP can make the GFP a more reliable reporter within a single construct.
Hi, i suppose GFP is expressed under CMV promoter and has additional WPRE sequence for stabilization whereas your gene of interest has a weaker promoter. I also observed such things and concluded that one can evaluate GFP expression only as a visible marker for infection efficiency but not as an amplitude indicator of your target gene expression. We are neighbours, so if you need help, just let me know ;)
GFP is easily visible and has a pretty long half-life (mRNA and protein). So its visible for a while, even when its not actively transcribed/translated any more.
If you get strong GFP expression probably either your construct has a problem (resequence it ) or Christian is probably right, check that your protein is not degraded by adding MG132 to the cells and trying to detect it.
I assume you are expressing the protein tagged and using a nice tag antibody for detection (V5, FLAG HA etc...?). If not then perhaps your protein specific Antibody is rubbish?
Another point to consider: if your pet protein is detrimental to the cells, they can easily methylate the CMV promoter, or employ a number of more exotic survival strategies. Did you check if the RNA is expressed?
A bicistronic pet-IRES-GFP or a self-cleaving polypeptide pet-P2A-GFP can make the GFP a more reliable reporter within a single construct.
Thanks guys, i did observed the target protein after immunostaining. Seems that the promoter is not strong enough (hPGK), now working to replace it with ubiquitin or CMV.
thanks Anna, i also heard about the problem of CMV, which most likely may be silenced in cells, like ES cells. Sounds a good idea to try EF-1.
Our target proteins are fluorescent calcium indicators, so not flexible for this part.
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