Transient expression vector - (Aug/03/2009 )
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.
chijing on Aug 3 2009, 08:46 PM said:
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.
Yes, as long as the cells have the gene.
cellcounter on Aug 4 2009, 11:24 PM said:
chijing on Aug 3 2009, 08:46 PM said:
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?
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.
bob1 on Aug 11 2009, 04:31 PM said:
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.
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.
genehunter on Aug 12 2009, 10:38 AM said:
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.
As far as I know, generating stable colonies by cotransfecting a selection vector with a target plasmid is routinely practiced.
cellcounter on Aug 13 2009, 12:50 AM said:
bob1 on Aug 11 2009, 04:31 PM said:
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??