Linearized or unlinearized plasmid for stable transfection? - (May/20/2005 )
This is a hard question to answer easily.
First: Pick a unique site (an enzyme that only cuts once) that does not disrupt you gene-of-interest, the promoter region or the poly-A site.
Two: Then ensure that the promoter to gene-of-interest to Poly-A site read-through is intact.
Most times, I have linerized after the poly-A site and before the promoter region but in some plasmids, this can be hard (i.e. no unique sites available).
hope this helps,
If I didn't cut the plasmid to do stable transfection, will the DNA randomly cut and recombinate with the chromosomal DNA?
If yes, what will happen if the gene of interest is cutted and integrate? Can it still be expressed?
if u study literature about introduction loxP sequences into genome you will find out that they always linearise before transfection since thiat might be a better substrate for stable transfectants. In our lab, we have had a case who used linearised DNA and she got stable clones, but I transfected my cells last week with circular DNA and I have good results , but I used phiC31 integarse that's why I have stable clones early, but other controls seem to be losing their plasmids. the BEST is to compare and let us know if you have time.
Just to add, i have used Cre/Lox recombination to produce stable cells via circular plasmid DNA and have succeeded in producing stable cells.
Have been passaging them for 20 close passages and the insert is still intact.
Havent tried linear plasmid DNA though.
Although the Cre/Lox recombination is an efficient method, the only set back is that you need to have a host cell that has integrated lox sites within its genome.
I have prepared stable transfectants using pCDNA vector (not linearized) , performed selection for 6 weeks in G418 and picked up isolated colonies. the strange thing I had was that clones with empty vector and those with vector harboring my insert are resistant to induction of apoptosis by my treatment as compaered to parental cell line. I mean the effect of my treatment is masked by that abnormally imparted apoptosis resistance to both types of clones irrespective of the insert. Most probably the unlinearized plasmid integrated into DNA in a focus that regulates apoptosis (this is the reply I received from the plasmid manufacturer technical support!!). I think I should have linearized the plasmid!!!!!!!!!!! now this wasted time and effort were gone for nothing!!!
The story is hard to sell, as integration sites will be random and you must collect more than one colonies for each line, right? I would suggest you to look into other reasons. Would the antibiotic that you used to select attribute to this phenotype change?
G418 is usually used for 4-6 weeks for selection. additionally this resistance happened with two different cancer cell lines. Actually I do not know the exact reason!!
I do not believe the answer from the technical support. both the linearized and unlinearized plasmid will integrated into chromosome randomly that means even you linearized your plasmids they still will integrated into so-called locus regulating apoptosis if you are really so lucky! As I understand, the advantage of using linearized plasmid will let your interested gene and other functional part including the region of promoter and expression cassette of selection marker etc integrate into chromosome without disturb.
I'm doing a side-by-side comparison of linearized vs. nonlinearized plasmid for stable transfection, using a pcDNA3 construct that hasn't been giving good expression levels for others in the lab.
One question: Does anybody treat the cut plasmid with phosphatase, or digest the overhangs, before transfecting with linearized plasmid?