Plasmid replication in mammalian cell systems - (Oct/14/2007 )
Hi,
I would like to ask whether anyone knows regarding Plasmid replication in mammalian cells.
I have been looking around but don't seem to be getting any luck.
My focus is on the establishment of stable cell lines.
My questions include:
1. Are plasmids maintained in the transfected mammalian cells? or do they immediately get integrated into the host genome upon antibiotic selection?
2. In the case of bacteria, an origin of replication is required for plasmids to be maintained within the bacterial host, but how does replication work in mammalian cells?
Thanks.
There are several ways to maintain/replicate a plasmid in a mammalian cell:
SV-40 large T and SV-40 replication origin is one possibility, but the plasmid tend to integrate into genome easily, and has high rate of recommbinantion over time. The replication rate is very high therefore host cells are in stress as a result. SV-40 T antigen is oncogenic. There is a work on mutation of SV-40 T-antigen to make it less oncogenic and safer replication protein.
EBV origin and EBNA is another pair for plasmid replication/maintainance at less copy numbers per cell. There are reports using EBV/mammalian replication origin hybrid system.
One group used mammalian S-MAR sequence with endogenous relication origin activity.
I hope this can provide you with some information for further study.
I would like to ask whether anyone knows regarding Plasmid replication in mammalian cells.
I have been looking around but don't seem to be getting any luck.
My focus is on the establishment of stable cell lines.
My questions include:
1. Are plasmids maintained in the transfected mammalian cells? or do they immediately get integrated into the host genome upon antibiotic selection?
In stable transfection DNA get integrated into the cell genome , thats why we select with antibody for several days but when we do transient transfection you will find your plasmid in episomal state*not maintained * and in some cells its integrated into genome and usually expression ceases after 2 or 4 days since episomal DNA is degraded by the cells .
2. In the case of bacteria, an origin of replication is required for plasmids to be maintained within the bacterial host, but how does replication work in mammalian cells?
Uaually plasmids dont have a duplication origin that works in eukariotes but they can work in procariots so thats why we can use bacteria to amplify plasmids while we cant use mammilians cells so its either the plasmid get integrated in the cell genome after selection in case of stable transfection or just be in the episomal state for 2 days enough to express ur transient protein in case of transient transfection.
hope this help youThanks.
Thanks very much for the detailed explaination and info.
It should be sufficient enough for me to further understand the basic of plasmid replication.