Efficient L929 transfection?

Dima Klenchin klenchin at macc.wisc.edu
Sat Sep 9 20:54:08 EST 1995


In article <christoph_reinhard-0709951752560001 at mainm3-005.chiron.com>,
   christoph_reinhard at cc.chiron.com (Christoph Reinhard) wrote:

>I am currently looking for a efficient transfection method for L929 cells.
>They seem to be very sensitive to lipofectamine and DEAE Dextran even at
>low concentrations. I wonder if anybody has experience with transfection
>of this cell type and could give me some hints. The results will be
>summarized and posted to the group.

Try electroporation. 
I have posted this thing couple times already. This is no fool proof
thing, but everything important is outlined. 

Fot stable transformation, for L929,3T3, CHO, Hela 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 
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+ (final Mg2+ is 2 mM!; do not add if you use 
	PBS that has already 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 have any questions or if this will help in any 
way, please email me - I’m curious to know results. 

- Dima





- Dima





will help. 



More information about the Methods mailing list