On 30 Jul 1996, Christa Baumstark-Khan wrote:
> Dear GFPlers
> I try to transfect CHO cells with the Clontech pGFP-N1 vector using a
> liposome transfection assay. So far we succeded in the experiment as
> under selection conditions (G418, 1.5 microgram per ml medium) we got
> realy nice colonies, surviving the procedure. So far, so good, but non
> (realy none!!!) of the colonies gives any shine of green fluorescence.
The CHO cells sometiimes are tricky for G418 selection. They tend to form
aggregates that survive the G418 selection. We found that in order to get
real colonies, one must split them, 24 hours after liposome transfection,
in 1 to 40 or more before the selection. The G418 selection needs several
rounds of DNA synthesis (cell divisions) to have the killing effect.
Another possibility is that the copy number of transfected plasmid is low
resulting in insufficient EGFP expression. I am sure it will be easy to
check this out by either PCR or some type of hybridization assays.
> We harvested such colonies and cells, which have not been transfected
> in order to measure fluorescence output in a spectrofluorimeter.
> Again, ther was not a glimpse of fluorescence. It was our 3rd
> transfection with the same result. Something is wrong, but what is it?
> Has anyone an idea?
>
_____________________________________________________________________________
Chris Lau, Ph.D. Tel: 415 476-8839
Division of Cell and Developmental Genetics FAX: 415 502-1613
Department of Medicine, VAMC-111C5
University of California, San Francisco E-mail : clau at itsa.ucsf.edu
4150 Clement Street
San Francisco, CA 94121
