Linear vs. Circular DNA

[Ian A. York]

In article <9411252122.AA26753 at calvin.jci.tju.edu> P_Norton at CALVIN.JCI.TJU.EDU (Dr. Pamela Norton) writes:
>>I would like to ask a question for which I am having a hard time finding 
>>an answer to.  I was told that it was not possible to generate stable 
>>mammalian cell lines with circular plasmid DNA, that it should be linearized 
>>first.  Is there a reason for this?
>>
>>David J. Austin, Ph.D.      
>>Department of Chemistry     austin at slsiris.harvard.edu
>
>        We have had reasonable success with stable transfection of circular
>DNAs into more than one mammalian cell type, but I suppose this may be
>cell-type specific. What cells do your sources who say that this can't be
>done use? Apparently, homologous recombination in transfect
>(electroporated) ES cells is greatly enhanced by prior linearization of the
>plasmid. Is this what you are referring to?

I can't speak from experience (never having tried to make stables with
circular DNA) but my understanding is that the linear DNA is *much* more
recombinogenic.  So as well as enhancing the efficiency of transfection
somewhat (if you're using electroporation - according to Maniatis) the DNA
that gets in has a better chance of incorporating into the genome.  This
is not (again, as I understand it) particularly cell-type specific, and 
we used to linearize DNA to make recombinant viruses as well for the same 
reason.  But circular DNA should work also, just less efficiently.  
	
	Ian

-- 
Ian York   (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-4328     Fax  (617)-632-2627