Luc- / GFP-Stability

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Andi
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Luc- / GFP-Stability

Hello,

I like to perform transient transfections with luciferase- and GFP-plasmids. Because of the time-protocol I have to meassure the activity of the reporter genes after 2 weeks. Has anyone an idea or experience with the stability of these vectors?

Thanx in advance,
Andi

Chin Fen Teo
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 Hi Andi,

 Hi Andi,

If you really have to measure the activity 2 weeks after transfection, I personally don't think it is a good idea to carry out the transfection in a transient manner. This is because there is very high chance that the untransfected cells will eventually catch up and dominate the cell population, thus you end up many cells without your plasmids and compromising your results.

Can you try to select for stable cell lines? Once you get the stable lines, you can always make many freeze down and thaw a vial when you need to repeat the experiments. And also, you can be sure that your luciferase and GFP are still there after this 2-week period of experimental time course...

Good luck...

Andi
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Hello Pippuri,

Hello Pippuri,

yes, you are right. The usage of stable transfected cells would be the better option, but I have to test several constructs before I can make stable transfected cells.
Luckily, the cells I have to use for these experiments stop dividing before the tranfection and they are "healthy" for the next two weeks. Do you think that reporter gene vectors are stable for 2 weeks in these cells?

Andi

Chin Fen Teo
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 Hi Andi,

 Hi Andi,

I don't think so.... :-(  Unless the plasmid somehow got integrate during the DNA replication cycle... 

But I may be wrong... If you have tried it out, I am very curious to know... :-)

Andi
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Hello Pippuri,

Hello Pippuri,

it will take some time, I have just started cloning. But I let you know my experience.

Andi