GFP and DsRed dual lenti construct - (Sep/07/2006 )
Hi There,
I have a big question. I am making lentivirus using 293 T cell and my lentivector construct is as follow:
MLC2v-GFP-EF1 alpha-DsRed. (noted: MLC2v and EF1 alpha are all pomoters and MLC2v should not be turned on in 293 T cell but only EF1 alpha).
Now the quesiton are two:
1.everytime my 293 T cells always have green (should not be shown) and red color after 24 hours of transfection?
2. Why everytime my 293 T cells is about to die after 24 hours transfection. Is DsRed toxic? or I just didn't culture well.
Many Thanks
Why do u say that MLC2v promoter will not work in 293T cells? As u c green fluorescence, u r having GFP expression so ur promoter should b working.
Dsred, is a tetramer, and aggreagtes and it can b toxic. Dsred2 is a better one than Dsred.
Remember, transfecting 293T for lentiviral production is itself toxic for cells.
BTW, do all the cells die or a fraction of it?
Dsred, is a tetramer, and aggreagtes and it can b toxic. Dsred2 is a better one than Dsred.
Remember, transfecting 293T for lentiviral production is itself toxic for cells.
BTW, do all the cells die or a fraction of it?
Thanks so much for the reply,
I said that MLC2v was not expected to be seen in 293 T because the myosin light chain-2v promoter (MLC2v) is a cardiac-specific promoter active in embryonic and adult ventricular myocardium, including human hearts. Of course, this confused us whether MLC2v also expressed in 293 T cells?
Secondly, we saw lots of 293 T cells dead after transfection but not all of them.
I will try your sugesstion of using Dsred2 instead, thank you so much.
Hope to hear more info from you
What part of the spectrum are you using to excitate DsRed? I have so far only used DsRed2 and even though clontech suggests that it can be efficiently excited at 488 nm, it's far from optimal.
Just yesterday I took some confocal pictures of DsRed2 tagged cells, excited at either 488 nm, 496 nm, 514 nm and 561 nm and only the 561 excitation gave a good signal (the other parameters were left identical).
293T cells on the other hand are good for expressing for protein expression, but bear in mind that DsRed2 excitation is really a bottleneck in most cases (and that DsRed2 takes double the time EGFP takes to be detected, has lower quantum yield etc...)
293 is HEK 293 - human embryonic kidney.
as its an embryonic cell line, there should b activity of the most promoters.