lentiviral overexpression of miRNAs
Posted 08 April 2010 - 06:15 AM
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??
Posted 08 April 2010 - 12:52 PM
Posted 08 April 2010 - 01:25 PM
Posted 08 April 2010 - 05:52 PM
Posted 08 April 2010 - 08:39 PM
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.
Posted 09 April 2010 - 01:59 AM
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?
Posted 09 April 2010 - 06:46 AM
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.
Edited by Functional Screens, 09 April 2010 - 06:48 AM.