4 replies to this topic

[Byung K. Min]

After transfection of lentivirus packaging genes + a gene of my interest into 293T cells, cells tended to grow more slowly than normal cells.
Besides, cells started to die at 24hptx, and most cells floated up at 72hptx.
A strange thing is medium turned to yellow despite low cell density.

Is it supposed to happen during virus production?

Can the virus production damage the host cells?

What could be the problem?

[Chaser]

You will observe cell death post-transfection.

What confluency do you aim for with your HEK the day prior to transfection?
What is the passage number of your HEK cells? I have had bad luck with virus production with higher passage number HEK. You might consider thawing down a fresh vial.

Edited by Chaser, 20 December 2010 - 07:04 AM.

[Byung K. Min]

I usually do transfection between 50-60% confluency.
I also tried on freshly thawed cells but got the same result...

[rmh999]

Byung K. Min, on 16 December 2010 - 08:54 PM, said:

After transfection of lentivirus packaging genes + a gene of my interest into 293T cells, cells tended to grow more slowly than normal cells.
Besides, cells started to die at 24hptx, and most cells floated up at 72hptx.
A strange thing is medium turned to yellow despite low cell density.

Is it supposed to happen during virus production?

Can the virus production damage the host cells?

What could be the problem?

What's the envelope protein?  VSV-G will cause syncytia to form between cells, killing them.

[UBClabbie]

What transfection reagent are you using? Some tend to be more toxic than others. I can recommend one with low toxicity if you'd like