Electroporation of Mammalian cells

[Dean Danner Lab]

In article 3eu at mercury.hgmp.mrc.ac.uk, jslingsb at crc.ac.uk (Mr. J.H. Slingsby) writes:
>We are interested in electroporating HepG2 cells.  Has anyone successfully 
>transfected constructs into these cells?  If so any guidance regarding 
>preparation of cells for electroporating, as well as which parameters
>(voltage, cpacitance, resistance) might be used will be appreciated.  
>There appears to be no reference in the literature to successful electro-
>poration of HepG2. If there are any refs. then this will suffice.
>
>Mr. J Slingsby and Dr. A K Vaishnaw
>Hammersmith Hospital
>London, U.K.
>E-mail jslingsb at uk.ac.mrc.hgmp
>


I have successfully electroporated HepG2s in a BioRad Gene Pulser.
	Protocol:
	-  Isolate cells.  Two confluent T150s will provide enough 
		cells for about 12 transfections.  Rinse twice
		in PBS, Trypsinize, wash with media (w/ 15% FBS) to 
		inactivate trypsin, resuspend in .2ml X (# of rxns +1).
	-  Set up reactions.  add 200 ul of cell suspension to cuvette
		containing 10 ug circular plasmid.  (I use 1 ug of 
		B-gal control, 2 ug of plasmid of interest and 7 ug
		of carrier plasmid)
	-  Zap.  My best conditions are: .25kV, 960uF.  Let sit in 
		cuvette for 15 min, add to 8ml fresh media (I use 
		60mm dishes for this).  This yields time constants from
		high 40's to mid 50's.
	-  Harvest after 36-48 hours

	Notes:  all of this is done at room temperature.
		I have only used this for transient transfections with 
			CAT plasmids, but I don't see why it wouldn't
			work for stables.
		Good luck.  If you need more details, e-mail me!

Tracy Sitler
Department of Genetics and Molecular Medicine
Emory University
djdlab at bimcore.emory.edu    or
tsitler at gmm.gen.emory.edu