Some of the information is from Addgene site. I thought it is better do a cut paste than type.

1. In a stable transfection, does the plasmid DNA always get integrated into the genome?

A stable transfection is used to create a population of cells that have fully and successfully incorporated foreign genetic material into their genomes. Unlike plasmids used for expression in yeast and bacteria, plasmids used for stable transfections rarely contain an ORI since the integrated DNA will be replicated as part of the genome. Because the foreign DNA becomes a permanent addition to the host genome, the cells will continually express the genetic traits of the foreign material and will subsequently pass it on to future generations. Stably transfected cells may be considered an entirely new cell line from that of the original parental cells.

In case the plasmid has a SV40 Origin of replication, cant the plasmid replicate on its own without getting integrated into the genome?

SV 40 origin of replication (ORI) is not sufficient for plasmid to replicate in mammalian cells. These ORIs, however, require additional components expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen (293E or 293T cells), allow for episomal amplification of plasmids containing the viral EBV or SV40 ORIs, respectively. The presence of these viral components greatly reduces the rate of plasmid dilution but does not guarantee 100% transfection efficiency.

Here plasmid is maintained as episome and transfection is considered as transient transfection.

2. In the case of integration into the genome, is it the entire plasmid that gets integrated or just a part of it? Once integrated into the genome, does the plasmid ori have a role in its replication or is it completely governed by the host replication machinery?

Depends. Some plasmids are constructed to introduce part of it (mostly based on recombinases and site specific recombination). Other wise entire plasmid is integrated.

Plasmid ORI (Procaryotic has no role in replication. SV 40 ORI may have some role if the cell lines express T antigen.

3. Does the sequence in the plasmid determine the integration site or is it a completely random process?

Some plasmids are engineered to get incorporated in a specific place. An interesting thing is you get  stable cell more efficiently  if the cell line has a integrated plasmid with sequence similar to your plasmid. (invitrogen FLPin cells). Another example is gene knockouts are generated using homologous recombination where more chances of recombination occurring  at specific place.

Otherwise integration is mostly a  random process

4. Once the cells are confirmed to stably express the protein of interest, how important is it to add the antibiotic (selection marker) during further culturing?

Not necessary to add it continuously. Make a stock with cells grown in the presence of antibiotic and cells from this stock can be used for a few (?) passages. in the absence of antibiotic.

You can refer

http://blog.addgene.org/plasmids-101-mammalian-vectors

Hope this information is useful

Thanks for the information, Mr.Rao. But if the DNA has been integrated into the genome, shouldnt it be stable in the absence of antibiotic? Is there something like in the absence of the antibiotic (selection pressure), the host may recognize it as foreign and result in its excision?

Hi Anjali,

Good question let us look at the population genetics of stable cell lines.

Host may not recognize the gene of interest or plasmid  as foreign and kick it out. In case, having  gene of interest is disadvantageous to host (say it is toxic to cell/ reduces the growth rate/ it interfere with expression of other genes due to position of integration) we may see selection against gene of interest.  There are more chances of accumulation of mutations in fast growing  Continuous cell lines and a cell which manged to get rid of expression of the gene of interest will be in an advantageous position and percent of its progeny in the population increases with time (passages?). This mutation could be a point mutation, deletion or insertion......  By growing  cells in the presence of antibiotic you are trying to prevent loss of expression due to gene excision (deletion mutants). There are more chances for maintenance of  the gene of interest  due to its proximity to antibiotic resistant gene.  Selection using antibiotic  may not have any effect on loss of expression due to point mutations in the gene of interest or its promoter region.  In other words growth in the presence of antibiotic will not guaranty expression of your gene of interest. 

I had been looking for some of the answers which are mentioned here. I had some problems with stable expression of a gene in Cholec1  cells. I performed a PCR on the extracted genomic DNA and confirmed the gene is integrated in the genome although, when I checked for expression it didn't see any. 

Thank you Anjali for asking this question and Panduranga for providing a succint reply. This is very helpful.

what is the mechanism behind the integration of foreign DNA into host genome permanently in case of stable transfection?

what is the mechanism behind the integration of foreign DNA into host genome permanently in case of stable transfection?

@tamali halder: I think probably due to the cells own DNA repair processes

Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.

Please see the following manuscript for details:

Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.

Hi all,

Thanks for this discussion. Although it was from long time, I am really interested to know : in case of the transformation using gene gun, what is the mechanism of the vector integration in the host genome!?