Eukaryotic (mammalian) replication origins are much more complex than bacterial ones. The chromosomes are so large that you need to have multiple origins in each chromosome. To ensure that each chromosome gets copied exactly once in each cell cycle, the associated proteins that control replication require phosphorylation by cyclin-dependent kinases to ensure that the build-up of the pre-replicative complex and the subsequent steps of DNA replication are each confined to the correct phase of the cell cycle and cannot occur concurrently ( https://en.wikipedia.org/wiki/Eukaryotic_DNA_replication )

In transient transfection, the plasmids do not get replicated, you just get whatever number of plasmids you managed to get into the cells, and are diluted out as the cells replicate. However, with low probabilities, plasmids will integrate into the host DNA and propagate along with the host DNA. You can increase this probability of integration through providing suitable sequences for homologous replication to produce relatively stable cell lines. If you want extrachromosomal DNA to get replicated within an eukaryotic cell, you normally go to viral vectors, e.g. Baculovirus in SF9 insect cells or Adenovirus-based constructs..

Dear Mishael al Bakar

differently to E.coli and S.cerevisae to date there is not a sequence that allow to maintaina and propagate the plasmids into mammalinan cells.

Stable clones could be generated performing the integration of the GOI sequence into the host DNA sequence. In standard cell lines, as HEL293, CHO, the integration of a single copy could be quite simple thanks to avaiability of some tecnologieis based on the presence in the genome host ofhomologiy recombinantion cassette (as the FLp-In system from Thermo).

Cell lines generated with this approach could be very usefull for express an antigen in the cell surface and use it for validate monoclonal antibodies by FACS, but not for recombinant protein expression in high yields, which require an high copy number.

as Annemarie Honegger told, in transient transfection (which is the common approach used for recombinant protien and antibody production in laboratory scale) the plasmids do not get replicated, you just get whatever number of plasmids you managed to get into the cells.

Some knock out cell lines as the GS Knock-Out were proved to be able to speed up the selection of a stable clone.

Article Attenuated glutamine synthetase as selection marker in CHO c...

ciao

Manuele

Manuele Martinelli & Annemarie Honegger Thank you guys so much!! That is really helpful.

So based on what I understand I have a question related to what you mentioned. If I'm constructing a plasmid vector for expression of mAb in CHO cells, does adding a viral ori such as SV40 make the vector a viral vector? similar case if I add an f1 phage ori

It's not as simple as just using the SV40 origin. In order to uncouple viral replication from the cell cycle of the host cell, viral genomes encode also several proteins needed for viral replication, e.g. the T-antigen of SV40 which is essential for replication.

Article SV40 DNA replication: From the A gene to a nanomachine

https://academic.oup.com/nar/article/27/2/426/1057041