Mammalian Electroporation?!?

[Dima Klenchin]

In article <3u31k6$218 at sifon.cc.mcgill.ca>,
   Graham Dellaire <popa0206 at PO-Box.McGill.CA> wrote:
>HAs anyone out there obtained an electroporation frequency
>better than 10-5 transformants/no# cells transformed???
>
Of course. In brief, for L,3T3, CHO 10e(-3) is readily achievable; for 
Jurkat, K562, primary fibroblasts 10e(-4) to 10e(-6) is more typical (I 
must say though that I’ve never had a goal to get the highest numbers). 

I’ve done my Ph.D. thesis on the mechanism of electrotransfection 
(a.k.a. electroporation). As I see a lot of electroporation-related 
questions on this group, I’ll summarize my experience and 
recommendations here 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? Electroporation 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, capasitance, 
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 do do it *with* DNA as DNA increases 
cell death (passing DNA enlarges electropores making some 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 works 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 then 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. 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. 
	Note: Ca2+ (100 uM and up) in electroporation medium is toxic 
for fibroblast-like cells but has no effect on lymphocyte-like cells. 

	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+ 
- add 0.5% Ficoll 400 is added;
(Do not autoclave! Sterylize by filtration). 

Ficoll at this concentration dramatically helps 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 has the same voltage and capacitors 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 I already have. Use noncut RNA-free plasmid at 50 
ug/ml and cells at 3x10e7 in 200 ul of the above electroporation medium. 
At highest capacitor available, optimize voltage to find 35% survival 
conditions, then repeat the same for transformation frequency/efficiency 
around the foltage found with smaller voltage increments. Than 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 to know results. 


Best of luck!

- Dima