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lentivirus infection - (Jan/22/2013 )

I am wondering if anybody has the same experience as me.
I generated lentivirus with a MSCV vector with GFP at the N terminal, encoding full length of my protein.
With the viral stocks, I infected multiple cancer cell lines. i could see green fluorescence under microscope and sorted the cells by flow cytometry. I did see lots of fluorescence after sorting. But the expression levels of the protein didn't change at all, tested by western blot.

I don't understand. If it is because of the cell toxicity of my protein.....? But why i could see so much fluorescence?
I am considering to switch another vector. But i still don't understand what happened to my protein. How could the GFP and my protein be separated?

If anybody can help me figure this out?

Many thanks.


What is between the two protein sequences: IRES or 2A peptide? Because the GFP is in front of your gene, it is not surprising that GFP has higher expression than your protein. You may need to consider a different vector.


Hi pcrman,
In the vector, my gene is actually in front of GFP, another word, GFP is at the N-terminal of my gene.