Myotubes transfection protocol wanted

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Dec 23 06:56:03 EST 1999


"Frank O. Fackelmayer" wrote:

> Wolfgang Schechinger wrote:
>
> > Here is speculative and non-proven theory:
> >
> > When you want an gene be expressed, the DNA should be located in the
> > nucleus. Thus, when you transfect cells, you must get the plasmid
> > into the nucleus. This should be very easy, when there's no nuclear
> > membrane present as it happens when cells divide.
> > This could be the reason wha some people see best transfection
> > results in non-quiescent cells.
> >
> > Wolfgang
>
> Hi Wolfgang,
> I have been looking around for explanations of how the transfected DNA
> enters the nucleus (because my scientific interest is nuclear
> architecture), but it seems no-one has ever really investigated this. It
> may be that DNA passively diffuses into the nucleus (even though it is
> quite big and may have problems to get through the pore), or there might
> be an active transport. I don´t think that mitotic breakdown of the
> nuclear envelope is sufficient for the DNA to enter the nucleus (how
> should a DNA molecule know where to get after mitosis?).  So, possibly,
> transfected DNA enters the nucleus bound to a protein that is actively
> transported into the nucleus. I wouldn´t expect that cells have a
> specialized "plasmid importer protein", but who knows? Possibly, a DNA
> binding protein takes it in "artificially" when it is imported into the
> nucleus after its synthesis.
>
> Wouldn´t it be interesting to perform experiments on that issue? Is
> anyone out there investigating these things? Or are there already papers
> on the subject that I may have missed by searching MedLine?
>
> Frank

Hi, its me again...
I just did a MedLine search again, and indeed there seem to people out
there who are intetrested in plasmid uptake. That´s an abstract of a JBC
paper I found:



Nuclear import of plasmid DNA in digitonin-permeabilized cells requires
both cytoplasmic factors and specific
 DNA sequences.
 Wilson GL, Dean BS, Wang G, Dean DA
 J Biol Chem 1999 Jul 30 274:31 22025-32

 Abstract
 Although much is known about the mechanisms of signal-mediated protein and
RNA nuclear import and export, little is understood
 concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and
14.4 kilobases were specifically labeled using a
 fluorescein-conjugated peptide nucleic acid clamp. The resulting
substrates were capable of gene expression and nuclear localization
 in microinjected cells in the absence of cell division. To elucidate the
requirements for plasmid nuclear import, a digitonin-permeabilized
 cell system was adapted to follow the nuclear localization of plasmids.
Nuclear import of labeled plasmid was time- and
 energy-dependent, was inhibited by the lectin wheat germ agglutinin, and
showed an absolute requirement for cytoplasmic extract.
 Addition of nuclear extract alone did not support plasmid nuclear import
but in combination with cytoplasm stimulated plasmid nuclear
 localization. Whereas addition of purified importin alpha, importin beta,
and RAN was sufficient to support protein nuclear import,
 plasmid nuclear import also required the addition of nuclear extract.
Finally, nuclear import of plasmid DNA was sequence-specific,
 requiring a region of the SV40 early promoter and enhancer. Taken
together, these results confirm and extend our findings in
 microinjected cells and support a protein-mediated mechanism for plasmid
nuclear import.

 MeSH
 Base Sequence, Cell Membrane Permeability, Cell Nucleus, Cytoplasm,
Digitonin, Fluoresceins, Hela Cells, Human, Microinjections,
 Nuclear Proteins, Peptide Nucleic Acids, Plasmids, Recombinant Proteins,
Support, Non-U.S. Gov't, Support, U.S. Gov't, P.H.S.,
 Transfection

 Author Address
 Department of Microbiology and Immunology, College of Medicine, University
of South Alabama, Mobile, Alabama 36688, USA.



Seems to be the information we have looked for, so now I´m going to read
that paper over the holidays.

: ) Frank







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