Linear vs. Circular DNA

[Dr. Pamela Norton]

>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?
>
> -Dave
>
>David J. Austin, Ph.D.
>Department of Chemistry aus...@slsiris.harvard.edu
>Harvard University
>Cambridge, MA 02138
>(617)495-8393
>(617)495-0751
>
Hi,

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 would be greatly interested in hearing from anyone who has had
better success transfecting linear versus circular DNA.

Pam
------------------------------------------------------------------------------
Pamela A. Norton, Ph.D. p_no...@lac.jci.tju.edu
Assistant Professor of Medicine
Thomas Jefferson University
1020 Locust Street, JAH 365
Philadelphia, PA 19107

[Ian A. York]

In article <941125212...@calvin.jci.tju.edu> P_No...@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 aus...@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 (yo...@mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-4328 Fax (617)-632-2627