Establishing stable cell lines...

Evans, Krista KEvans at lifetech.com
Mon Sep 18 12:38:25 EST 2000


Before even transfecting cells for stable clones, you need to establish a
killing curve for your particular cell line (don't assume). You should also
perform a transient transfection to optimize your lipid and DNA.  You need
to maximize transfection efficiency as only a low percentage of cells will
integrate the plasmid.
The protocol that I have used with success is to transfect your CHO cells as
you would for a transient assay with the neo encoding plasmid (use controls
of no plasmid and a plasmid of similar size with a reporter gene).  The day
after transfection, make split ratios of various sizes (depending on how
fast the cells normally grow) for all transfected wells and controls.  The
day after the cells are split, apply media containing the appropriate amount
of G418 to your wells (you should also have duplicate wells that do not get
any G418 as controls for cell killing/growth).  Do NOT allow your cell
colonies to grow together (this is where a large split ratio is important).
Allow at least 14 days for transient expression to diminish before selecting
and counting clones or staining for reporter assay.
CHO cells are notorious for migrating on the plate...it is very difficult to
isolate a single clone colony.  Simply changing the media causes the cells
to detach, migrate, and reattach in a different spot (I've seen some lovely
almost spiral colony patterns due to this phenomena).  Be VERY careful when
changing media with these cells.  Good luck.
Krista Evans
Life Technologies Division of Invitrogen

>Sept. 14, 2000 writes:
>Dear All 
>I am trying to establish stable mammalian cell lines expressing a
transfected gene by G418 selection in CHO cells. 
>I treat the transfected cells with G418 @ 400ug/ml (I assume this is this
enough ?) but don't see extensive cell death or gain much >expression after
2 weeks selection. 
>Could the problem be that I use Lipofectamine 2000 and get so much DNA into
the cells that too many of them remain resistant to >the G418 ? Might a less
efficient transfection protocol like calcium phosphate work better in the
long run ? 
>Thanks in advance for any advice. 
>JJ 


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