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Lentiviral transduction, no target protein expression and lost other reporter


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#1 HCC

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Posted 27 July 2011 - 07:37 AM

Hi,
I ordered a PC-3M-luc-C6 cell line from Caliper Life Sciences, which expresses firefly luciferase. I transduced the cells with renilla luciferase lentiviral particles. I grew up colonies that were resistant to Puromycin. However, when I tested the cells' firefly and renilla luciferase activity, I found that there was no renilla luciferase activity and I had lost the firefly luciferase activity. I'd appreciate any help with regards to what may have happened and how I could fix it.
Thank you!

#2 powertoeki

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Posted 12 August 2011 - 11:12 PM

I went to PC-3M-luc-C6 cell lines from Caliper life science and according to this website, cells are selected using Zeocin. SO when you were tranducing the cells with renilla luciferase using lentivral particles, did you select cells for zeocin along with selecting cells with puromycin? If you don't provide cells with zeocin while transduced or after being transduced, they may have lose firefly luciferase expression during this time...

Also... I believe in order for cells to express antibiotic resistance to those selection, for ~1 day after lentiviral particles, incubate cells with no selection (puromycin). However, from what I see from your cells you, I would incubate cells with zeocin in order to keep PC-3M-Luc-C6 cells to express firefly luciferase. Some people don't start selecting until like 2-4days later in order to increase 'their chance'.

Do you have proper positive control for lentiviral particle trasduction efficiency?

You also might have to do a curve for puromycin selection. Make sure to select cells with both, zeocin and puromycin.

Good luck!!

Edited by powertoeki, 12 August 2011 - 11:19 PM.





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