stable transfection

Martin Kennedy cytogen at chmeds.ac.nz
Thu Nov 12 00:10:32 EST 1992


In article <40729 at sdcc12.ucsd.edu>, wsun at jeeves.ucsd.edu (Fiberman) writes:
> Hello Netter,
> 
> I am going to use the pRc/CMV vector from Invitrogen to express a cDNA
> in HELA cells.  I want to select for the transfected cells with
> neomycin resistance and establish a stably transfected cell line
> expressing my clone.  Can anyone out there recommend a good reference,
> or give some helpful tips on how to proceed?  Any suggestions
> would be appreciated.
> 
> -fm

First of all, determine the G418 sensitivity of Hela cells with your batch of
G418 and in your culture system.  Culturing in medium containing from 100ug/ml
up to 1mg/ml of G418 should reveal the best lowest conc. that causes killing. 
G418 takes a few days to kill cells.

Then make a large (preferably cesium, but other prep methods work) plasmid
prep, and linearize your construct anywhere in the vector sequences.  Then
transfect by whatever method works best for Hela cells, and place cells under
selection about 24 or 48 hours later.  You should get resistant colonies in
about a week or two.  You can pick these manually in several ways.  I use a
flame drawn, bent pasteur pipette, with mouth suction device (and cotton wool
filter).  Replace the medium in your plates with PBS.  Suck up some trypsin in
the pipette, and (with a binoc microscope) carefully eject the trypsin over and
around the colony to be picked. Scratch of the colony, and suck up the cells. 
Transfer to trypsin, until single cells obtained, and then plate back into
selective medium on 24-well plates.> 

Some useful references are:

Michael Kriegler:  Gene transfer and expression; a laboratory manual. Stockton
Press, 1990.

E.J.Murray (ed):  Gene transfer and expression protocols. Methods in Molecular
Biology Vol 7.  Humana Press 1991.

	(Especially Chapter 19, R.F.Santerre et al., Use of vectors to confer
resistance to antibiotics G418 and hygromycin in stably transfected cell lines;
pp245-256).

Hope this is useful,

Cheers,
Martin



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