Verified Plasmids(via digestion) > Transfection and Visible GFP > GFP de - (Oct/22/2013 )
Hello All Dear Researchers,
I have very very big problem. I have been working on a project till 1,5 years. Last week I faced with really dramatic result. I am so confused. Please help me about this problem.
We have a project about JAK2 V617F Mutation and its effects about endothelia cells. We took plasmids from a lab/group which works in USA. Vectors are 5 plasmids: pCDF1-GFP(no JAK2 gene), pCDF1-JAK2 WT-GFP, pCDF1-JAK2 MUT V617F-GFP and structural and envelope (vsvg) vector. (FIV-Based Lentiviral Vectors)
I transformed this plasmids. Colony selection. Miniprep. Maxiprep. Enzyme Digestion. All these steps are hurdled. Then I made transfection with these plasmids/vector (lipofectamine 2000 protocol followed). 293 T cell line used. After 72 hours later transfection I saw GFP expression under fluorescence microscopy. Followed ultracentrifuge protocol and obtained (expected) lentiviruses.
Using these viruses HUVEC cell lines infected. These cells analyzed via Flow Cytometry and GFP included cell detect. Infection efficiency were really nice (rates change % 60-90). BUT I COULD NOT SEE ANY GFP UNDER FLUORESCENCE MİCROSCOPY (even confocal).
Then I sorted cells via Flow Cytometry but interestingly GFP (+) cells also have no GFP under microscopy. I checked result with pcr (after genomic dna isolation) but there were no true gel image/band.
Please Help me!!!
Where is my problem I do not exactly !!!
Are plasmids problems(mutation) ? Cell line (293 T/HEK) ? Arent plasmids integrated to genome really?
Is a no one else ?
Please help :(