Currently, I am doing knockdown of certain genes by shRNA technique. We use lenti-virus for this process. The problem is that we are getting low titre. The experimental procedure is:
Day 1: Seed 7 x 10^6 HEK-293T cells in a 10 cm dish.
Day2: Transfection by using Trans-IT transfection reagent. (Cell confluency=60-70%) (Plasmid DNA 7.5 ug)
Day3: Harvest the supernatant and concentrate to get virus particle using PEG-it™ Virus Precipitation Solution (5×).
I usually get titre of 5 x10^4 to 5 x 10^5 IU/mL)
I've encountered a similar thread previously. I've linked it below, maybe the answers there will be useful to you
https://www.researchgate.net/post/How_can_I_increase_my_lentivirus_titre_and_infectivity_particularly_for_transducing_SH-SY5Y_cells
Two points I'd make in addition to the ones in the other thread:
1) Use more DNA. For each 10cm dish, I use 20ug of total plasmid DNA mix.
2) I've noticed that concentrated virus containing PEG can result in fusion of your target cells resulting in syncytia formation and eventually cell death. If you can adapt to a concentration protocol that avoids the use of additional reagents (ultracentrifugation is my preferred method but it depends on if you have access to an ultracentrifuge)
Govinda is correct, PEG can sometimes cause problems. What LV plasmid are you using? Do you have a map? How are you assessing your titre?
Dear Govinda Sharma and Jill Muhling,
Thank you for the suggestion.
The very first thing I did is that I increased the concentration of plasmids (from 7.5 ug to 15 ug) with all other experimental condition intact. I tried in 2 dishes (10 cm) and the viral titre was improved with high plasmid DNA. However, I need to confirm this result with repetitive experiments.
I use pCAG-HIVgp and pCMV-VSV-G-RSV-REV plamids for Lenti virus production.
I assess the titre by Real time PCR.( https://www.abmgood.com/High-Titer-Lentivirus-Calculation.html)
OK, how about your transfer (shRNA or gene of interest) plasmid? Do you have a map for that?
There is a lenti focused webinar coming up that might address your concern and help answer any additional questions.
Title: Knockdown, Knockout, Validate: Lentivirus delivers payload in vitro and in vivo Date: Wednesday, May 16, 2018 Duration: 60 minutes Times: 10:00 AM (CST) Speaker: @Christy Hoffmann
What you will learn: Lentivirus biosafety features and best handling practices Flexible customization options to fit your needs Techniques to incorporate lentivirus into your gene studies Insight from a leading Lenti manufacturing expert
Register here: https://www.sigmaaldrich.com/life-science/learning-center/customer-education/lentivirus.html
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