We obtained pLKO5-EFS-GFP from addgene (Ben Ebert Lab) which contains U6-gRNA-EF1s-GFP. Using transient transfection, we got very good transfection. With virus, the GFP expression is extremely weak although 50-70% cells were infected. The virus was made by our core that offers good service and has good reputation. We are puzzled... Has anyone used the pLKO5 based lenti? Anyone had the same problem?
Was this with a non-integrating vector? The fluorescence intensity is usually much lower per MOI compared to an integrating vector. What cells did you transduce, and at what dose?
What's the cell line you had transduced?
What's your protocol of transduction?
You can have a look here to improve your transduction protocol:
http://www.geg-tech.com/knowledge/protocols
Nico
I assume you used the same cell line for both transfection and transduction. Then I think the issue is related to MOI and transduction efficiency. For example how much virus (titer? MOI?) you added to the cells? Did you ad Polybrene? Did you use spinoculation? It may give you 5-10 folds (or even more) difference in GFP intensity.
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