GFP transfected HeLa, advice?

Wang Min johnson_lab at qm.yale.edu
Tue Sep 26 18:44:31 EST 1995


Any help on this would be greatly appreciated...
   pRAY-1 is GFP inserted into pDNA3 (CMV promoter-driven GFP), that I
obtained from GIBCO/BRL.  I transfected transiently pRAY-1 into HeLa cells
and saw nothing by FACS or fluorescence microscopy (the transfections, as
measured by a second reporter gene, were good).  So I made stables and
picked 12 G418-resistant clones, still nothing over background
(autofluorescence) on the Fluorescein, Rhodamine, or Texas Red (?) FACS
channels.  
   It seems that the plasmid must be these, since the clones are resistant
(parallel mock transfectants died), and the CMV promoter is very strong in
these cells...  
   This constuct (pRAY-1) has been used in mammalian neuron cells (I
think), so the coding sequence (codon usage) should be good enough.  Are
there any "tricks" to get the protein to fluoresce or to detect it?
   Thanks for any advice (or commiseration!)
   -Dave



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