transfection of JEG-3 cells

D.K. dk at no.email.thankstospam.net
Wed Sep 29 21:42:21 EST 2004


On 30 Sep 2004 02:06:58 +0100, walterschick at astound.net ("Walter Schick -
BioSepCo") wrote:

>Agree with D.K.  An Ambion poster recommends square wave electroporation for
>primary and hard to transfect cells because one needs the higher cell
>viability results.

I think this advice is most likely wrong. From practical point of 
view my limited experience (long ago) with primary skin fibroblasts 
was that exponents worked better than squares. From theoretical point 
of view this is something to be expected because "electroporation" 
is a two-stage process: first electropores are created by high 
intensity electric field and second DNA is electrophoresed into
pores by the electric field (Biophys. J., 60:804-811, 1991 and 
Biophys J., 63:1320-1327, 1992; these conclusions have been 
confirmed many times on various systems). The electophoresis part 
can be carried with less damage to the cells at lower but longer 
pulses - the exponent's tail does this nicely. So, if there is 
a problem with viability that extreme that with long pulses 
the relatively low voltage necessary is not creating pores 
efficiently, one is better off opting for a smaller capacitor 
and increased V/cm and still using exponential discharge rather
than switching to square pulses. 

Of course, ideal if a two-pulse square system (see the second
reference above), but those that are commercially available are 
much more expensive and take more to optimize. 

>Bacteria and fast growing mammalian cells can be grown
>back, even with low cell viability from chemical transfectants. However,
>electroporation will work with all eukaryotic cells, so why not use it for
>every line?  

With all cells! From mycoplasma and chloroplasts through spermatozoids
and yeast to the fish eggs and whole drosophila embryos. 

>However, if you don't have an electroporator, 

These days, it is virtually guaranteed that there is someone
nearby that have it. 

DK

>try looking up some of the
>hundred or so papers that have sucessfully transfected with chemicals or
>lipids.  You may have to try something else than ca-phosphate.
>
>W Schick
>----- Original Message ----- 
>From: "D.K." <no.email at thanks.to.spam.net>
>To: <methods at hgmp.mrc.ac.uk>
>Sent: Wednesday, September 29, 2004 3:53 PM
>Subject: Re: transfection of JEG-3 cells
>
>
>> In article <11b853f5.0409260711.df01deb at posting.google.com>, 
>> naomi.coslovsky at weizmann.ac.il (nc) wrote:
>>>Need advise on transfecting JEG-3 cells:
>>>I didn't succeed to transfect my JEG-3 cells with the modified
>>>Ca-phospate method (with BES not HEPES). my regents are probably fine
>>>as I can transfect other cell lines I use. Any advise how to improve
>>>the transfection is wellcomed.
>>
>> Use electroporation. It always works.
>>
>> DK
>> 
>
>---




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