In article <32FB2C89.6EA2 at sheffield.ac.uk>,
Kevin A. Mulcahy <K.Mulcahy at sheffield.ac.uk> wrote:
>>I am having some problems with transfection of EBV-transformed B cell
>lines. The method I am using is electroporation - I have had also been
>successfully transfected by other groups) but I don't seem able to
>succeed in generating stable transfectants from other EBV-transformed B
>cell lines. Thus, I was wondering if anyone doing such transfections
>could give me some help.
Kevin, we only have succeeded in transfecting a few of the many EBV
lines that we have in our lab. If you need to transfect a specific line,
you may be out of luck. I have found that you are unlikely to recover cells
unless you start with a cell line that grows reasonably vigorously (splits
at least 1:4 twice a week). Nevertheless, I will tell you what we do.
>Firstly, could you please let me know how you do your transfections, for
>(a) growth conditions of the cells (e.g. density and freshness of
>medium) prior to electroporation;
We grow cells in RPMI 1640 supplemented with 10% FCS. I put my
flasks on their sides in order to ensure adequate oxygenation. The night
before I transfect, the cells are typically just over 1 million/ml. I
then split 1:1.
>(b) harvesting, washing and preparation of cells for electroporation
>(including electroporation buffer/medium);
>(c) electroporation parameters;
I wash cells once in PBS, then resuspend at 10 - 15 million per ml
in PBS or "cytomix" (check Biotechniques? for the latter reagent, I don't
have the original reference). I place 250 ul of the cells in a 0.4 mm cu-
vette. To this I add 10 - 20 ug DNA in water, not exceeding 10% of the
final volume. I have used either a BTX or a Bio-Rad electroporation appar-
atus with similar results. I always use 960 uF capacitance and 200-300 V.
>(d) treatment and culture of cells following electroporation and time
>point of introducing antibiotic selection.
After the cells have been zapped, I dilute them to 1.5 ml in RPMI
plus 25% FCS and place them on a 48-well plate. Two days later I count them.
In cultures that will eventually grow out, I count between 20% and 60% of
the initial live cell concentration. I don't bother trying to count dead
cells -- it's very inaccurate. Sometimes the cells have already turned the
medium yellow by this time. At this point I remove half the medium and over-
lay it with 25% FCS, G418-containing medium at double strength. Thereafter,
I use 1X medium with 25% FCS. Over the course of the first week, the cells
may turn the medium yellow once more before the massive die-off occurs. Feed
them if this happens.
Now wait. Wait some more. Four weeks is not unreasonable, though
six weeks probably is. Eventually you will see an increase in live cell
density. It's O.K. to resuspend the cells to get a look at them. If you
don't, and they're lying on the bottom of the well in a pile of dead cell
debris, it might be hard for you to see them. The medium may turn yellow
again. Overlay the cells with fresh medium if you're worried about low
density. Otherwise, you can start splitting. Split moderately at first,
harder once you know you've recovered something. You can also cut back
to 10% FCS once you have 10 ml or so. However, since you're using an
episomal vector, you will always need to use some G418.
>Secondly, I did manage to transfect another EBV-transformed B cell line
>with my cDNA (the vector I am using is pREP9 from Invitrogen, which
>confers resistance to G418 and possesses both the EBV origin of
>replication and the EBNA-1 gene for episomal maintenance) but the cells
>grew very slowly in selection medium (only doubling about once a week,
>if that!). In addition, when I tried cryopreserving them and then
>thawing-out a vial they did not survive (they were thawed-out directly
>into selection medium). Also, I have not been able to stably transfect
>them with the vector alone (i.e without insert cDNA) and I was therefore
>wondering if the concentration of the G418 was too high?
One difference in our procedure: when we transfect episomally, we
use the pHEBO vector, which confers hygromycin resistance. When we work
with G418, we use the integrating vector pBJ1-neo (Lin and Davis, Nature
1991). I don't know how this will affect G418 doses.
>Therefore, what criteria do people use for determining the appropriate
>concentration of antibiotic? I had performed a titration of the drug in
>24-well plate format using 200-1200µg/ml (100µg/ml steps) and found that
>the minimal dose required to kill all of the cells in 2 weeks was
>700µg/ml and therefore 700µg/ml was used for selection of transfectants
>(700µg/ml was also used for routine sub-culture of the transfecants).
>However, at 400, 500 and 600µg/ml the number of viable cells in the
>24-well plate were 65/276, 9/148 and 1/89, respectively. Would it have
>been more appropriate to have gone for say 600µg/ml (or less) or would
>it not be worth risking the possible proliferation of the "1 viable cell
>in every 89 cells" (which may die given longer selection periods)?
We count cells over the course of one week, not two. We place cells
in 25% FCS-enriched RPMI 1640 medium with varying amounts of G418. Some of
the cultures will actually grow for a few days before they start to die back.
If they should turn the medium yellow, I replace half the medium with fresh
stuff -- or, in the case of a positive control, I split it. I find that the
test points needn't be closer together than a factor of 1.5 to 2. In other
words, testing 400 ug/ml, 500 ug/ml, and 600 ug/ml is nitpicking. My typical
battery of concentrations is 50 ug/ml, 100 ug/ml, 200 ug/ml, 400 ug/ml, 800
ug/ml, 1.2 mg/ml, 1.8 mg/ml, and 3.0 mg/ml. All the cells I tried will grow
in the lowest concentration, and all cells will die in the highest concentra-
tion. As the G418 is added from a 100 mg/ml stock solution containing NaCl
and HEPES, I make up a solution containing just these components, without
G418. I test a 1:33 dilution (equivalent to my 3.0 mg/ml G418 solution) on
the cells. One of my cell lines was inhibited by HEPES and NaCl alone!
The goal is to find the lowest concentration at which the *net*
growth rate of untransfected cells is just slightly negative over the course
of the week. I typically count *live cells only* on day 0, day 2, day 4,
and day 7. I then graph the results and eyeball a straight line through
the four points, because each day you count on the hemacytometer, you will
get variable results. The cells never resuspend quite the same way. Almost
all of the *human* EBV lines I have tested go negative at 1.2 mg/ml or 1.8
mg/ml. Three of the non-human lines I have tested were sensitive at 200
ug/ml. I was also unable to transfect these.
In my opinion, having live cells around after a week is not a bad
thing. Indeed, with EBV lines which sustain themselves by autocrine loops,
a certain minimal density of live cells is probably essential. People who
want to *clone* EBV cells by limiting dilution usually have to resort to
cytokine supplementation and/or irradiated PBMC feeder cells. Just let the
untransfected cells die off slowly and be replaced by transfected cells.
>Many thanks for your help,
Unique ID : Ladasky, John Joseph Jr.
Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
Location : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords : immunology, music, running, Green