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Transient expression vector


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#1 chijing

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Posted 03 August 2009 - 08:46 PM

I would like to know can a transient expression vector with a gene of interest cloned in it, create cell line stably expressing the non-endogenous protein after transfection.

Edited by chijing, 03 August 2009 - 09:04 PM.


#2 cellcounter

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Posted 04 August 2009 - 07:24 AM

I would like to know can a transient expression vector with a gene of interest cloned in it, create cell line stably expressing the non-endogenous protein after transfection.

There is no such thing as transient or stable expression vector. Any vector that integrates in the genome, can act as a stable expression vector.

The difference is, stable integration is a rare event, so if your vector has a selection pressure (Neo, Hygro, GFP), you can easily identify the stable clones, so these vectors are used for making stable cell lines. You can also use them for transient expression. When you transiently express a vector, without any selection, that rare stable integrant is hard to find.

#3 scolix

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Posted 04 August 2009 - 07:46 AM

Yes, as long as the cells have the gene.

#4 chijing

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Posted 10 August 2009 - 08:18 PM

I would like to know can a transient expression vector with a gene of interest cloned in it, create cell line stably expressing the non-endogenous protein after transfection.

There is no such thing as transient or stable expression vector. Any vector that integrates in the genome, can act as a stable expression vector.

The difference is, stable integration is a rare event, so if your vector has a selection pressure (Neo, Hygro, GFP), you can easily identify the stable clones, so these vectors are used for making stable cell lines. You can also use them for transient expression. When you transiently express a vector, without any selection, that rare stable integrant is hard to find.



I was transfecting my cells using a mammalian expression vector pCMVSport6 which carries the gene of interest and a contransfect vector pBABE-Hygro which contains the selection marker. AFter 10days of hygromycin selection, I did a pooling of the cell colonies left and checked on the overexpression of protein of interest. Is it possible to obtain stably transfected cells in this way?

#5 bob1

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Posted 11 August 2009 - 04:31 PM

Not really, as the plasmid that doesn't have a selection marker on it (pCMVsport) should be lost and the pBABE kept. Though if the pBABE vector has an integration site in it, and the pCMV recombines, then it is possible.

#6 cellcounter

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Posted 12 August 2009 - 08:50 AM

Not really, as the plasmid that doesn't have a selection marker on it (pCMVsport) should be lost and the pBABE kept. Though if the pBABE vector has an integration site in it, and the pCMV recombines, then it is possible.

I agree with bob1. The co-transfectant would allow you to select which cells have been transfected with your primary plasmid, but that does not guarantee or allow you to select the cells which have integration of primary plasmid. So, chances are very rare that you will get a stably transfected cell line this way. You need to clone the Hygro cassette in pCMVsport.

#7 genehunter

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Posted 12 August 2009 - 09:38 AM

Although it is less defined than the approach of cloning into pBABE, it is possible. If you used a large ratio of the target vector (pCMVsport) vs the selection vector (pBABE), like 10 to 1, you have better odds. Plasmid integration is more or less a random event, if pBABE integrates, so should the pCMVsport. Unless your gene has negative effect on cell growth, it will stay.

You can select/verify target gene positive clonies from the stable clonies.

#8 cellcounter

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Posted 12 August 2009 - 11:28 AM

Although it is less defined than the approach of cloning into pBABE, it is possible. If you used a large ratio of the target vector (pCMVsport) vs the selection vector (pBABE), like 10 to 1, you have better odds. Plasmid integration is more or less a random event, if pBABE integrates, so should the pCMVsport. Unless your gene has negative effect on cell growth, it will stay.

You can select/verify target gene positive clonies from the stable clonies.

More than random, it is rare event. Two different plasmids getting integrated in the same cells is not a high possibility. The only way this method can help is that you at least select the cells which were transfection prone. Integration is a toss.

#9 genehunter

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Posted 13 August 2009 - 08:58 AM

As far as I know, generating stable colonies by cotransfecting a selection vector with a target plasmid is routinely practiced.

#10 chijing

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Posted 13 August 2009 - 09:52 PM

Not really, as the plasmid that doesn't have a selection marker on it (pCMVsport) should be lost and the pBABE kept. Though if the pBABE vector has an integration site in it, and the pCMV recombines, then it is possible.

I agree with bob1. The co-transfectant would allow you to select which cells have been transfected with your primary plasmid, but that does not guarantee or allow you to select the cells which have integration of primary plasmid. So, chances are very rare that you will get a stably transfected cell line this way. You need to clone the Hygro cassette in pCMVsport.


How can i clone the hygro cassette into the pCMVsport backbone??

#11 chijing

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Posted 13 August 2009 - 10:05 PM

Although it is less defined than the approach of cloning into pBABE, it is possible. If you used a large ratio of the target vector (pCMVsport) vs the selection vector (pBABE), like 10 to 1, you have better odds. Plasmid integration is more or less a random event, if pBABE integrates, so should the pCMVsport. Unless your gene has negative effect on cell growth, it will stay.

You can select/verify target gene positive clonies from the stable clonies.



Usually i use 30ug of plasmid DNA for transfection but in a 1:1 ratio (15ug from each pBabe and pCMV).
Is this the problem why i'm not getting cells overexpressing the target protein?

#12 genehunter

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Posted 16 August 2009 - 08:51 AM

Usually you will have different colonies that express at different levels. This is because they vary dramatically in chromosomal integration site and the copy number integrated. You need to do a limited dilution, get the colonies, and screen for individual colonie for protein expression levels.




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