</biogeek talk. Please image a funny story about a cat with knit boxers>
There is some sort of flurescent protein which is toxic to cells in high concentrations… I just can’t remember what it is! Arrg I read some paper about it!
As for our GFP cell line we’re trying to stabalized…
I think the problem is in the cloning techniques… The person who made the clone had a hard time trying to transform bacteria with it… The Kan resistance gene isn’t working well at all. We’re getting like 5 colonies w/ 10uL of a 1:1 dilution of transformed JM109s. And this is at 30ug/mL of Kanomycin (spelling?).
As for the cells themselves… We’re getting nothing by FACS… Hell we got more positive cells for GFP in our isotype control :(. And we’re using the same vector (just swapped in Kan resistance for the Carb resistance) we use with cell surface expression constructs :/.
It’s not a down regulation over time… nor is it upregulation by time. We’re using selection media for the cells, the cells aren’t dying off which means they have the plasmid we grew. We know it’s the right plasmid since we grew up a new batch on the Kan+ LB-agar plates…
So my guess is that damn plasmid got cloned wrong. But then again I just went over the sequences and they look good. So maybe it’s an issue with the vast majority of the plasmid DNA kicked out the GFP insert but the primers for sequences are picking up only the plasmids w/ the insert… So maybe we only have 1% of the plasmids w/ GFP and Kan resistance the rest only having Kan resistance.
But then again, it’s not my project and I’m only a tech and I only do virus work:)
WHOOT! (It’s not something I have to fix!)
Then again, the real problem could be that we just got a new electroporator for mamalian/bacteria cells and nobody knows how to use it right yet and the other electroporator is only for bactiera 
As for the glowing genital moneys… The hypothetical goal was to make a chimeric replication defecient HIV-luciferase virus targeting to the mucosal cells in the reproductive tract. But yeah, I can see how using the specific promoter would take care of it too…
Maybe if I was to make it so the viral intergration site was for a specific gene only activated in germ cell production… That way only germ cells that would be infected would produce luciferase.
Hrrm, maybe not replication defecient virus maybe just a non pathogenic virus… cause it’d be funny to make a new SDT that didn’t damage cells, but did make your genitals glow.
It’d make proving adultery a LOT easier for divorces!
“Baliff (spelling!) turn off the light and Mr and Mrs Smith, drop your pants.”
“As you can see Mr Smith’s penis is glowing, yet his wife is unaffected. Clearly he had to have contracted the CRorex virus from SOMEONE other than his wife. I rest my case.”
Man that was a lot of biogeek talk. To those of you who read that the take home message is: wouldn’t a glow in the dark SDT be funny? It wouldn’t kill you, or cause and physical harm other than after you got it you wouldn’t need a flashlight to read a magazine under the covers
Or a night light.
Hell, you wouldn’t have to turn on the lights to pee anymore.
No gonna do it.