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Transfection of a non human gene into human cell line


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#1 sanjiun

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Posted 26 December 2012 - 06:35 PM

I am helping my colleague to transfect a viral gene into human cell line to study the effect of the viral gene in human.
Both of us tried many times but the transfection failed. Cells died (plasmid control, gene-of-interest and untransfected cells) after adding antibiotic to select.

Initially she thought it could be the transfection system/ kit (Tet-On 3G System from Clontech) problem.
But the system works fine for me (transfect and overexpressed other human gene), therefore she asked me to help her with the viral protein. This time, transfection failed. Again.

Could the problem be the viral protein itself? Is it really that hard to transfect?
Anyone tried transfecting viral protein? What kind of protocol/ system you are using?

#2 bob1

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Posted 26 December 2012 - 07:30 PM

So the controls are fine for the transfection or do they also die? There can be problems with transfection and/or overexpression of any gene, particularly if it interferes with some critical cellular process or has a toxic product.

As it is a viral gene, you could check whether infection at the same time as infection rescues the viability problem (only works if your transfected gene is tagged and detectable).

I routinely transfect with PEI using a 3:1 ratio of ul PEI:ug DNA. Fugene also works well for me, but I find that lipofectamine2000 causes more cell death than other reagents.




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