lentiviral overexpression of miRNAs - (Apr/08/2010 )

hi,

i have cloned the precusor sequence of my miRNAs of interest into pCDH-CMV-EF1-Puro-GFP (SBI) and performed lentiviral infection. The infection efficiecies were so different in the same cell line. One was fine (let's say A) while the efficiency of the other was very low (let's say . I measured the plasmid concentration several times to ensure the amounts of plasmids I used were similar. I also prepared the midi again, but I got the same result. Any ideas??

Samantha

Did you titer your lentivirus and use the same MOI in your transduction experiment? Packaging efficiency might not necessarily be the same for different constructs...?

Sorry, I am a bit confused. You said infection, but you mentioned your plasmid concentrations are not much different. Also, I wonder how you determined infection efficiencies.

I didnt measure the MOI for my experiment. However, when I transfected the plasmids into 293FT packaging cells, most of the cells got GFP fluorescence for both plasmids A and B. Using the supernatant to infect my target cells, only a small amount of cells got GFP fluorescence for supernatant from plasmid A, compared to that from plasmid B.

In order to rule out the miRNA precursor sequence interferes the lentiviral packaging/production, my suggestion is to isolate total DNA @ 12-24 hr post-transduction, then perform real-time PCR to determine the lenti cDNA copy number (you can make a primer set targeting GFP).
If the lenti cDNA copy numbers are quite different between A and B, the we might say something wrong with lentiviral production.
If the lenti cDNA copy numbers are not much different, then there is a possibility that the miRNA precursor sequence affects the GFP expressed by EF1a promoter.

Thanks Functional Screens.

So do you mean I should extract genomic DNA from my target cells 12-24hrs post-transduction, followed by PCR to determine the copy number of GFP that has been integrated into the genome? IF the amounts of GFP that has been integrated between the supernatants from two plasmids are similar, that means my precusor sequence affects the GFP expression. On the other hand, if the amounts are different, there is something wrong with the lentiviral production. Is this right?

Samantha

Yes, you're right.
Please note, for HIV/lenti infection: @12hr late-RT reaction reaches the peak; @24hr 2-LTR circle reaches the peak; @24hr integration reaches the peak. So, you are detecting most of late-RT products plus some LTR-circle and integrated cDNA. However, it gives you an idea how many copies of lentiviral genome get into your model cells.
Please wash your cells before extraction to remove plasmids carried over from your lentiviral transduction.
Good luck.