Mechanistics of Electroporation - Nucleus DNA

[Andreas "Mega" Stuermer]

Hi guys, 

A question came up that I found interesting. 

Electroporation is a standard method in vitro and in vivo. 

The DNA swims around next to the cells, and then the electrical pulses drag the DNA through the membrane. And the membranes might also have developed pores from the shock. 

Why isn't the DNA from the nucleus pulled out of the cell? Its backbone is negatively charged as well. I assume that the cell as a whole has a charge (the neagtively charged membrane builds a faraday-like cage??) and thus the cell is deformed, but the charge within the cell is homogenous? 

I mean, electroporation would not work if the nuclear DNA would just be dragged out of the cell so I assume that is what heppens. 

Would be cool to see if anyone has deeper insight

[Nathan McCorkle]

Short answer is, electroporation opens the cell, electrophoresis moves your DNA in, but also any other ions too. This applies similarly for the inner contents, they may move out... Given enough time. I think the key is concentration of exogenous DNA is great enough that it's just outside of the cell when the pores form, and your open time is just enough for the length of the molecule to move inside before the pores close.

As for the nucleus, I'm not sure I've seen anything on what happens to the nucleus, whether pores also form in there. It's an interesting question that I'm sure I could get sucked into a good day or more of searching and digging through references.

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[Tom De Medts]

At https://en.wikipedia.org/wiki/Electroporation#Gene_electroporation

See under sub-header: Gene electroporation,

 also copy / pasted below:

Application of electric pulses of sufficient strength to the cell causes an increase in the trans-membrane potential difference, which provokes the membrane destabilization. Cell membrane permeability is increased and otherwise nonpermeant molecules enter the cell.^[49][50] Although the mechanisms of gene electrotransfer are not yet fully understood, it was shown that the introduction of DNA only occurs in the part of the membrane facing the cathode and that several steps are needed for successful transfection: electrophoretic migration of DNA towards the cell, DNA insertion into the membrane, translocation across the membrane, migration of DNA towards the nucleus, transfer of DNA across the nuclear envelope and finally gene expression.^[51] There are a number of factors that can influence the efficiency of gene electrotransfer, such as: temperature, parameters of electric pulses, DNA concentration, electroporation buffer used, cell size and the ability of cells to express transfected genes.^[52] In in vivo gene electrotransfer also DNA diffusion through extracellular matrix, properties of tissue and overall tissue conductivity are crucial.^[53]

In eukaryotic cells, the nuclear DNA is inside the nuclear envelope, traffic through which is tightly regulated by the nuclear pore complex which is generally too small for nuclear DNA to pass through,

especially because such DNA is packaged into chromatin fibers wrapped around nucleosomes etc. So the cargo is too large to pass through, from inside the nucleus, outside into the cytoplasm, through the narrow nuclear pores.

That is one reason why electroporation works best in one direction, and not in both :)

At the outer cell membrane, external DNA that passes through, into the cytoplasm, I suspect is not just leaking through, but transported either actively or passively, by a pump or channel respectively.

There must be selectivity at various boundaries, especially at the cell membrane and nuclear membrane that makes this much more uni-directional that one would imagine based just on principles of electricity and ion transport.

Note that generally the concentration of specific ions is unequal across various membranes, for various biological reasons (energy generation, prevent toxicity etc.). 

This is possible due to selectivity of membrane pumps and channels. 

So the same property of membrane selectivity must operate even during electroporation, albeit less effectively, due to the electrical zap during electroporation creating a brief imbalance.

Cheers,

TdM

 

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[Bryan Jones]

It's got to come down to size. Chromosomes are 10s-100s of megabases in Eukaryotes and single-digit megabases in microbes. The biggest stuff I've ever seen electroporated in is 10s of kilobases, and even then efficiency starts to drop pretty quick over 10 kB. It does take a bit of fine-tuning of electroporation conditions for each cell-type to get the DNA in without letting too much essential stuff (like genomic DNA) out and killing the cells. 

--Bryan Jones

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