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I am reading through the ENCODE papers, which is taking me well out of my comfort zone in terms of modern laboratory techniques. At the risk of asking a question which may well be thoroughly answered somewhere else on the internet, I was hoping for a brief explanation of nuclear transfection.

In Whiteld et all, they describe the direct assay of transcription factor binding site efficacy in human cells. To do this, they transfect a luciferase plasmid, presumably into the nucleus of the cell (because they subsequently observe that the luciferase plasmid is transcribed).

Reading through Materials and Methods and getting to the supplementary file, they disclosed their transfection protocol. The relevant reagants appear to be:

FuGene 6

a nonliposomal formulation designed to transfect plasmid DNA into a wide variety of cell lines with high efficiency and low toxicity.

(From Promega's website.)

Lipofectamine LTX

efficient reagent for plasmid delivery and protein expression

(From Invitrogen's website.)

PLUS reagent

PLUS™ Reagent is used in conjunction with transfection reagents, such as Lipofectamine™, to enhance transfection in adherent cell lines.

(From Invitrogen's website.)

Apparently, the protocol is to combine the DNA plasmid with transfection reagent, wait a few minutes, and then add in the cells, and incubate at 37ºC for 24 hours. That's all that is required to get the plasmid into the nucleus.

I have consulted Wikipedia and Strachan and Read's Human Molecular Genetics 4th edition, but been unable to answer my question about this technique.

I think I have a pretty clear understanding of how these reagents are able to get through the cell's lipid membrane, but I don't have a similar understanding of how the DNA then passes to the nucleus.

In Human Molecular Genetics 4th edition, I can find only one paragraph about plasmid transfer across a nuclear membrane, page 704: "Transport of plasmid DNA to the nucleus of non-dividing cells is very inefficient because the plasmid DNA often cannot enter nuclear membrane pores. Various methods can be used to faciliate nuclear entry such as conjugating specific DNA sequences or protein sequences (nuclear localization seuqences) that are known to facilitate nuclear entry, or compacting the DNA to a small enough size to pass through the nuclear pores."

But nothing in the materials and methods section of Whiteld et. all mentions any of these techniques. I'm sure they would have mentioned if their plasmid included a DNA conjugation sequence, so that can't be it. Perhaps these reagents implicitly include some sort of DNA compaction polycation, such as PEG-CK30?

Isn't the cytoplasm an extremely hostile place for DNA plasmids? Shouldn't they just flail around in the cytoplasm until they are digested?

Or perhaps these vectors sneak into the nucleus during mitosis? (This is a theory that just occurred to me when contemplating the "non-dividing" qualification in the Strachan quote.)

Since the question isn't addressed in the paper, I can only assume the answer is elementary and something that a professional cell biologist would be expected to know. I've run out of obvious next steps for finding the answer, so I beg pardon in advance for asking it.

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2 Answers 2

up vote 7 down vote accepted

My attempt to find an answer has suggested that no-one knows how the DNA gets into the nucleus.

This fairly recent paper reports attempts to track the pathway of DNA entry and transfer to the nucleus.

Le Bihan et al. (2010) Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale. Nucl. Acids Res. 39:1595-1609

The technique used was transmission electron microscopy. Complexes of plasmid DNA + cationic lipids (lipoplexes) were labelled with silica-based nanoparticles to allow visualization.

The authors describe a transfection pathway with five steps:

(i) binding of the lipoplexes to the cell surface;
(ii) entry of the lipoplexes into the cells mainly via endocytosis (a direct fusion with cell membrane cannot be excluded);
(iii) DNA release into the cytosol;
(iv) transport through the cytosol; and
(v) entry of DNA into the nucleus and nuclear transcription.

The authors were able to follow each of the first four steps but:

our data show nanoparticles close to, but not within, the nucleus.

They speculate about what this might mean, but they do not refer to any previously published data. My conclusion is that the answer to your question is, as I said at the beginning of my answer: no-one knows how the transfected DNA enters the nucleus.

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Thanks Alan! I'm upvoting this for the nice article summary, but leaving the question unanswered in case someone else can comment specifically on Whiteld or shed some more light. –  masonk Sep 14 '12 at 16:05

This is a question that keeps coming up now and then when PhDs or post-docs discuss transfection. A probable answer for which I do not have supporting literature is this: If you have the transfection complex in the medium while the cells are dividing, it is possible that they can enter the nucleus following mitosis when the nuclear membrane is still forming, thus giving it access to the transciption mechanism in the nucleus.

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Hello and welcome to the site. Is there no supporting literature, or do you just not have it at hand? If the latter, then you are encouraged to add citations. –  kmm Oct 9 '13 at 13:35

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