In article <421b69$k5l at lilith.uab.es>, Jordi Barquinero
<jbarquin at gm.iro.es> wrote:
> Peter (& other fluorescent -bright- netters):
> I have read your message. We are currently trying to develop a retroviral
> vector producing cell line that carries the GFP gene for marking studies
> with hematopoietic cells. Our bicistronic construct contains the neoR
> gene under the LTR and the GFP gene driven by the TK promoter.
TK gene is likely to be too weak. By FACS you need at least 5000
molecules of GFP to observe (assume 50% quantum efficiency) IF you have
autofluorescence compensation set.
So far, we
> have transfected several cell lines (with lipofectin), including several
> ecotropic and amphotropic packaging cell lines. After G418 selection,
> some lines express GFP but others do not. For instance, HeLa and GPE86
> do, but NIH3T3 or PA317 don't.
tk promoter is weak probably
Although transfected packaging cell lines
> express GFP, no functional retroviral vectors are produced. Has anyone
> had success with a GFP retroviral vector?
Yes, MFG vector using mutant GFPs for brighter/faster fluorescence. Driven
off of LTR.