linear or supercoiled plasmid for creating stable cell line - (Dec/27/2006 )
Hi Gang,
I've made stable cell lines in the past and have always used a supercoiled plamid. Some of these have worked for varying degrees over the years. One thing I have noticed though is that even though these were selected as monoclonal cell lines, the activity of the cells (based on the gene I transfected in) changes over the years.
Is this due to the plasmid being maintained as an episome rather than integration into the chromosome? If it is episomal, how does the gene get transcribed anyway?
I know if I linearize my plasmid prior to transfection, I will get a much lower transfection rate, but it is supposed to increase the chances of chromosomal integration. Is this true?
Will a cell line be more stable over time if I linearize it and hope for chromosomal integration? Is it possible for the DNA machineary to cut pieces off the plasmid prior to integration into the genome?
My vector is pIRES (bicistronic expression) my insert is 1.5kB.
When transfection is successful, the phenotype of the cell changes, so it is easy to moniter success rates.
Thanks
I've made stable cell lines in the past and have always used a supercoiled plamid. Some of these have worked for varying degrees over the years. One thing I have noticed though is that even though these were selected as monoclonal cell lines, the activity of the cells (based on the gene I transfected in) changes over the years.
Is this due to the plasmid being maintained as an episome rather than integration into the chromosome? If it is episomal, how does the gene get transcribed anyway?
I know if I linearize my plasmid prior to transfection, I will get a much lower transfection rate, but it is supposed to increase the chances of chromosomal integration. Is this true?
Will a cell line be more stable over time if I linearize it and hope for chromosomal integration? Is it possible for the DNA machineary to cut pieces off the plasmid prior to integration into the genome?
My vector is pIRES (bicistronic expression) my insert is 1.5kB.
When transfection is successful, the phenotype of the cell changes, so it is easy to moniter success rates.
Thanks
So, you have quite a few issues here, Bluescript. If you want to use linear plasmid, you have to optimize your transfection with linear plasmid, not supercoiled (it's a different configuration when the DNA condenses). Linear plasmid doesn't increase your chances of integration, but does keep the gene of interest from breaking when recombination occurs.
The only way your circular plasmid is maintained episomally is if it uses the EBNA system (or possibly SV40 large T antigen, but I'm not sure about that one).
When you say they were selected as monoclonal cell lines, does that mean your line started from one cell? If not, you've got a population of cells that have been integrated in various sites in the chromosome. And yes, cells can shut off expression if they're not happy with the product they're being forced to make (even from a monoclonal population).
Stability of expression has more to do with where the plasmid integrated. But culture conditions can also affect your population of cells and their expression. Changing media can cause the expression to drop or disappear from an otherwise stable line.
In short, I would do a limited dilution sub-cloning several times to get a monoclonal stable expressing line after transfection. It's tedious and time consuming, but it works.