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How to control cloned cDNA expression level in cells?


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8 replies to this topic

#1 jiro_killua

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Posted 27 February 2009 - 10:16 AM

If I want to overexpress a gene in a cell, by transfection (using lipo2k)

And I don't want to have too high expression of it (cos that might not tell me the physiologic effect of the gene)

Can I monitor the dosage of the protein in the cell by changing to amount of plasmid I mix with lipo2k?

My question is basically this:

If I change the amount of plasmid I add, which of the following would most likely change:

1. Transfection efficiency?
2. Expression level of my target gene?
3. Nothing?

#2 scolix

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Posted 28 February 2009 - 08:02 AM

Try making stable cell lines to get higher expression which can be a little over the base line. With transient transfection one may have for eg. 1000x over expression.

If you change the amount of plasmid for transfection, you can have a change in transfection efficiency and also expression level.

#3 jiro_killua

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Posted 28 February 2009 - 09:46 PM

stable cell line is not an option

I'm doing primary cell culture, and I've never passage the cell (not able to)

any thoughts?

#4 stardust

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Posted 01 March 2009 - 03:20 AM

Hi,

What kind of a promoter are you using? If it is a viral promoter the expression will be very high...maybe choose a weak promoter?

Stardust

#5 scolix

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Posted 01 March 2009 - 06:09 AM

stable cell line is not an option

I'm doing primary cell culture, and I've never passage the cell (not able to)

any thoughts?


you could infect cells with viral particles, (Lentivirus, AAV) coding for the trangene and use a weak promoter to get a low level expression.

#6 jiro_killua

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Posted 01 March 2009 - 07:06 AM

Hi,

What kind of a promoter are you using? If it is a viral promoter the expression will be very high...maybe choose a weak promoter?

Stardust



now is CMV promoter....

#7 stardust

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Posted 01 March 2009 - 02:16 PM

CMV is very strong, I don't think you will ever get "physiological" levels of protein expression even if you had the option for making a stable line...can't recommend a promoter now since I don't know which cells you are using...

Stardust

#8 jiro_killua

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Posted 02 March 2009 - 04:35 PM

CMV is very strong, I don't think you will ever get "physiological" levels of protein expression even if you had the option for making a stable line...can't recommend a promoter now since I don't know which cells you are using...

Stardust



I'm using hepatocytes isolated from fetal mice

Thanks a lot

#9 stardust

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Posted 06 March 2009 - 05:18 AM

we often use PGK promoter (phosphoglycerate kinase) in our cells, it is strong as well but not as strong as CMV...otherwise google for weak promoters, many people will use nonviral promoters...or maybe find a promoter that is specific for hepatocytes..

Stardust




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