Efficient L929 transfection?
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.
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 Ive never
had a goal to get the highest numbers).
Ive 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, Ill
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, Id say, cells 1x10e7-5x10e7/ml and DNA 20-
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 isnt 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
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. Dont 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
7. Standard PBS (sorry, dont 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
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 dont however, see
how any other system that has the same voltage and
capacitors could be worse.
If I were to set good electroporation protocol for my needs,
Id 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 - Im curious to know results.
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