Transfection of Sf9/Sf21 Cells

polk at molbiol.ox.ac.uk polk at molbiol.ox.ac.uk
Mon Jun 6 05:08:59 EST 1994


In article <garry.23.0011B4AB at menzies.su.edu.au>, garry at menzies.su.edu.au (Garry Myers) writes:
> I've recently become the proud owner of the CLONTECH BacPAK expression
> system ;).  The protocol accompanying the kit suggests using Lipofectin(tm)
> to transfect the Sf21 cells with recombinant virus.  I'm interested to hear 
> any thoughts or opinions from baculoviral-minded individuals on other 
> methods of transfecting Sf9/Sf21 cells, particularly electroporation.
> ___________________________________________________________________
> Garry Myers                       Menzies School of Health Research
> Molecular Genetics Unit           garry at menzies.su.edu.au
> ___________________________________________________________________


Hi there,


Sorry for the other abortive post.

Electroporation works fine for Sf21 cells but efficiency depends on several 
parameters. However, production of recombinant baculoviruses by cotransfection
of transfer vectors and linearised 'wild type' baculovirus DNA into Sf cells
is very efficient and much simpler using lipofectin ( 5 microliters for 35 mm 
dishes is enough).

If you wish to try electroporation: we've used-

	using a BRL electroporator (trademark etc) at 1600 uF
	with 0.4 cm electrode seperation and 1 ml cuvettes

	resuspend cells in chilled HEPES buffered glucose saline
	and pulse to give 60-80% cell viability by trypan exclusion.
	DNA uptake is maximal at 65-70% and is quite poor outside 
	60-80%. Also keep cell conc. < 3 million per ml as excess cell fusion
	results/ and low DNA uptake.

	550 volts/cm fieldstrength with the above conditions worked for us

	in our hands electroporation was 100 times better than
	calcium phosphate and 3-5 times better than lipofectin
	for introducing viral DNA into Sf21 cells

I can supply more details if required.

Best of luck

Ian

Ian Polkinghorne    POLK at molbiol.ox.ac.uk         NOTE: All comments are mine
NERC Institute of Virology                        and do not represent NERC, 
& Environmental Microbiology                      Oxford Uni, IVEM, or the
Mansfield Rd Oxford OX1 3SR  UK                   QLD DPI.. etc and so forth
(Just another Aussie)



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