For all you, electroporation fans! (Was: Re: Electro-transformation of mammalian cells)

Dima Klenchin klenchin at macc.wisc.edu
Mon Apr 22 08:48:43 EST 1996


In article <3175639C.37C2 at erlangen.netsurf.de>, Heinz-Juergen Schaefers 
<h-j.schaefers at erlangen.netsurf.de> wrote:
#Hi!
#
#I am looking for a protocol for transformation of plasmids (4-8 
#kbp) into mammalian cells (rat kidney cells) via electroporation.
#If anyone has experience with that, please provide me with the details.

Below is FSA (Frequently Sent Article) that I feel might be helpful. 

*****************************************************************************

I have done my Ph.D. thesis on the mechanism of 
electrotransfection (a.k.a. electroporation). When I realized how many 
Qs conserning it appear in this group, I summarized my experience and 
recommendations based both on practical and theoretical 
considerations. 

The mechanism is electrophoretically driven DNA insertion 
through electropores (see Klenchin et al., Biophys. J., 1991 
and Sukharev et al., ibid, 1992). Hence, the condition:
        1. Pulse should be low amplitude/long duration exponent 
(pores created are larger and live longer; long nonporative 
and less damaging "tail" works for electrophoresis). 
Practical sense: use the highest capacitor available and 
find optimal voltage (see below). 
        
The probability for the DNA to get to the pore increases 
with increasing both cell and DNA concentration. => 
        2. Increase both. It all depends on exact situation but 
generally, I'd say, cells 1x10e7-5x10e7/ml and DNA 20-100 ug/ml. 

        An important distinction: are you interested in total 
number of transformants obtained (*efficiency*) or you need 
*frequency* of transformation to be high? Pores formation  
and, in part, DNA input depend on voltage ~ exponentially, 
whereas concomitant cell death dependence isn't that sharp. 
=> For the former, one might want to go for very harsh 
pulsing conditions killing 90+% of cells and high cell/DNA 
concentrations. For the latter, you need to find a 
compromise between DNA uptake and cell death. Practically 
speaking: 

        3. fix electroporation medium, suspension volume, 
capacitance, choose cell and DNA concentrations and 
"titrate" voltage. Conditions where 25-50% of cells survive 
(by plating and counting, not by Trypan Blue! T.B. 
underestimates death dramatically) should cover window of 
optimal frequency. It is important to do it *with* DNA as 
DNA increases cell death (passing DNA enlarges electropores 
making more of them irreversible). 

        4. Room temperature works better in 90+% cases. 
        5. It depends, but generally supercoiled and circular 
plasmids work better for transient, while cut plasmids work
better for stable transfections. Effects here could be small or 
large depending on cell line and plasmid itself. 
        6. Don't bother with more than one pulse. 
        7. No prepulse incubation of DNA is necessary.

Electroporation medium is a tricky issue. Effect on the 
level of electricity is simple: in low salt the pulse is 
much longer than in high salt (but in sucrose the same 
amplitude/duration gives lower frequency). All other effects 
are on the level of cell survival. One can spend life 
optimizing components of the buffer. Currently, most people
use cell culture media (MEM, RPMI, etc. - with or without 5-10% serum). 
        Here is my approach to the problem. I found the buffer that 
appeared to work equally well for a variety of cell types (including even 
Drosophila cell line) and, in my hands, better than 
everything else described in the literature (YMMV). 
        Note: Ca2+ (100 uM and up) in electroporation medium is toxic

        7. Standard PBS (sorry, don't remember protocol) is 
modified in the following way: 

- HEPES is used instead of phosphate (it is frequently 
        referred to as HBS);
- K+ and Na+ are swapped (e.i., the solution is 
        "intracellular" - high K+, low Na+);
- add 2 mM Mg2+ (if you PBS does not already have 2 mM MgCl2; 
        if it does, don't; *final* Mg2+ should be 2 mM)
- 0.5% Ficoll 400 is added;
        (Do not autoclave! Sterylize by filtration). 

Ficoll at this concentration dramatically helps cells to survive 
(why? - mystery) thus enabling to increase voltage and/or 
duration. We used to call this buffer TrakhoDim (TD, after 
Dima). 

        8. While no postpulse incubation is necessary, 5-10 min 
after pulse do not affect survival significantly. Cells, 
however, should not be diluted right away into warm culture 
medium. Use rt medium and let pores reseal for ~ 20 min 
before placing cells in CO2 incubator. 

        9. Bio-Rad pulser is very good. I don't however, see 
how any other system that offers similar voltage and 
capacitance range could be worse. 

Bottom line: 
~~~~~~~~~~~~
If I were to set good electroporation protocol for my needs, 
I'd do the following: 

Stick to the pulser you already have. Use noncut RNA-free 
plasmid at 50 ug/ml and cells at 3x10e7 in 200 ul of the 
electroporation medium of your choice. At highest capacitor 
available, optimize voltage to find 35% survival conditions, 
then repeat the same for transformation frequency/efficiency 
around the voltage found with smaller voltage increments. 
Then stick to the best voltage for the given cell line. 
Repeat the same for every cell line separately. 

If you guys have any questions or if this will help in any 
way, please email me - I'm curious.  

- Dima





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