In article <01bbbfd6$9e62c400$21746a8d at meb_493a.cvrc.mcw.edu>,
Joe Miano <jmiano at post.its.mcw.edu> wrote:
>>If a transgene is stably integrated in a cell line through selection with
>G418, why is continued G418 necessary with clonally-derived cells? It
>seems that once the gene is integrated and cloned, no further G418 would be
>necessary, n'est ce pas?
I don't think that anyone knows the explanation for sure, but all
integration events are apparently not created equal. Some DNA constructs
seem to integrate in a semi-functional fashion, in which one gene is ex-
pressed but not the other. So it is possible to obtain cells that are
drug-resistant but not expressing the gene of interest. These cells often
have a growth advantage over the transfected cells in the absence of drug;
they're not burdened by expressing the foreign gene. Keeping the drug
around levels the playing field a bit. But, even in this case the trans-
fectant might have a slight disadvantage. So freeze down aliquots early!
The fraction of cells that are successfully expressing both the
drug resistance marker and the gene of interest is variable, and may depend
on the cell type transfected, the plasmid used, or the gene you want to ex-
press. Most of the folks in our lab transfect MHC class I cell surface
proteins into mammalian cell lines. They analyze their transfectants by
flow cytometry using fluorescent monoclonal antibodies. They find that only
about 30% to 70% of the primary transfected cells are expressing the class I
molecule. Positive cells are then sorted on a FACS machine.
My observations are a bit different. I use different cell lines than
my colleagues, but the same plasmid. My insert is beta-2 microglobulin rather
than MHC class I. I find that all of the cells I recover from the primary
transfection express at least a little beta-2m. I sort anyway to obtain
a purified population of highly-expressing cells.
Unique ID : Ladasky, John Joseph Jr.
Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
Location : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords : immunology, music, running, Green