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Troubleshooting on lentiviral biosystem

lentivirus overexpression noGFP nofluorescence

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#1 Darlidada



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Posted 18 May 2015 - 06:43 PM

Hello everybody , 


Considering how your previous reply were useful, i'm back with a new troubleshooting .

I wanted to do an overexpression of my protein of interest in min6 cells. because of the low transfection efficienty in these cells, i used a lentiviral biosytsem

I bought a plasmid from Origen : it was plasmid including fusion protein of my protein and turboGFP, after many checking , i removed the insert part my fusion protein and turboGFPand i did the insertion in trip lentiviral vector ( the final size was 13 000 kda). I did the transduction, the titration, transfection .... then i checked for the mrna expression of my protein of interest and i get only 5 fold increase, i decided to check the turboGFP signal : NOTHING !!!!!!


I've no explanation for this, please helppp, it drives me crazy

I should add some precisions: 

- at the same time, i did the same experiment with another viral construction : my protein of interest into the trip lentiviral vector ( without turboGFP) and in this case i observed about 30 fold of change at mrna level

- another crazy thing is that , i did the dose response of death : i used an antibiotic of selection : neomycin, so these cells survived that's mean that they integrated the lentiviral vector , ????blink.png

- I still have this tiny increase in mrna level of my protein of interest ??

- I didn't check for tGFP before my experiment because of the stupid microscope, it was impossible to make the settings at the P2 laboratory.


How it's possible ? all your hypothesis are welcome

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