Plasmid replication - Plasmid replication (Mar/04/2009 )

My question is
every one of us use several plasmids for transfection into human cells for ex into 293t or hela cells. i would like to know how does plasmid replicate in the human cells.since they are not integrated into host genome but still they replicate, i would like to understand the mechanism.

thank you very much for helping me understand this question

Who said that they replicate?

Hi there,

it depends a lot on your plasmid. If you transfect a plamids with mammalian replication origin in cells, the plasmid might replicate.

For example, plasmids carrying the SV40 origin can replicate in 293T cells since they have the large T antigen See here

Another example for a replication origin in mammalian cells is EBNA from eppstein Barr virus.

"Normal" plasmids without any eukaryotic or mammalian viral origin of replication can not replicate in the cells, the cells divide, the plasmmid is lost. Unless of course you have a selection marker on the plasmid (Puro, Neo, BS,...) then under selection the plasmid will integrate into the genome of the cell and "replicate" with the chromosome they integrated on.

Stardust

stardust on Mar 5 2009, 11:35 AM said:

Hi Stardust, can you explain how the selection markers will allow the plasmid to integrate into the genome?

Do you mean pressure is on the cells to keep producing the protein that metabolises the particular antibitoic (and thus the plasmid that has the genes for it) and so only cells that keep the plasmid in their nucleus or cytoplasm will continue to grow in the presence of the antibiotic but that the plasmid does not actually integrate?

Hi,

since in general plasmids can't replicate at all in mammalian cells only cells can survive which have integrated the plasimd into their genome because otherwise you loose all the plasmids during cell division. Lets say 1 cells recieved 4 copies of the plasmid by transfection and the plasmid can' replicate then in the next cell division 1 cell makes 2 cells each of which contain 2 plasmid copies, next division 2 cells make 4 cells 1 plasmid each, next division only half of the cells contain plasmid and so the plasmid s lost with divisions. So, the plasmid which carries a selection marker can not replicate and if the cells "want" to be resistant it has to integrate the plasmid in its genome and replicate the foreign dna with its genome since now the plasmid is part of the genome. otherwise the cells die. So, unless you have a mammalian or viral origin of replication on the plasmid the plasmid HAS TO integrate to give resistance to the selection agent.

Stardust

so if the vector has mammalian ori there is no need to integrate?hm....didn't know, thanks for the info. do these vectors need to be inside nucleus in order to replicate into the new dividing cells? how many sister vectors can be produced approximately? am i asking a stupid question?...lol

can you give us some commercial examples of such vectors?

I want to insert my PCR product into a vector and make loads of it. do I need to transform it into E.Coli to replicate? or if the vector has mammalian ori then I can simply transfect it into human cancer cells, and later lyse the cells and extract the plasmid?...the question is how to separate it from the genomic DNA? run on agarose gel and then cut the plasmid band? am i talking offtopic? sorry... ...I mostly do protein work and i'm new to cloning.

SV40 origin does not work very efficiently in human cells, even in the presence of SV-40 T antigen.
In monkey cells, it replicates too much and kills host cells.
in human cells, I would say the plasmid still ends up with either lost or integrated to the genome.

stardust on Mar 6 2009, 05:14 AM said:

Curtis on Mar 6 2009, 06:24 PM said:

thank you again tea-test,

I thought because of the size of cells they can hold up many plasmids at once although doubling time is 24 hrs.