Help...cell transfected with empty vector(control) don't retain fluorescence - PIRES2 (Jun/06/2008 )
plasmid : PIRES2-EGFP
I transfected cells with PIRES2-EGFP carrying mutant gene, wildtype gene, or empty vector (but should still express EGFP), grow them in G418 for selection
i have repeated experiments several times..
each time：
cells transfected with the empty vector doesn't form any good fluorescent colonies...there are only very small and weakly fluorescent colonies.. after i pick the colony and innoculate them into new wells, the fluorescence just disappear.. maybe becoz the fluorescence are too weak..and the transfection is not stable
but cells transfected with mutant and wildtype form bright green fluorescent colonies..wonder what's the problem...
i think cells transfected with empty vector should have least impact on the cells...but it's not the case
-coolku-
QUOTE (coolku @ Jun 6 2008, 11:21 AM)
plasmid : PIRES2-EGFP
I transfected cells with PIRES2-EGFP carrying mutant gene, wildtype gene, or empty vector (but should still express EGFP), grow them in G418 for selection
i have repeated experiments several times..
each time：
cells transfected with the empty vector doesn't form any good fluorescent colonies...there are only very small and weakly fluorescent colonies.. after i pick the colony and innoculate them into new wells, the fluorescence just disappear.. maybe becoz the fluorescence are too weak..and the transfection is not stable
but cells transfected with mutant and wildtype form bright green fluorescent colonies..wonder what's the problem...
i think cells transfected with empty vector should have least impact on the cells...but it's not the case
I transfected cells with PIRES2-EGFP carrying mutant gene, wildtype gene, or empty vector (but should still express EGFP), grow them in G418 for selection
i have repeated experiments several times..
each time：
cells transfected with the empty vector doesn't form any good fluorescent colonies...there are only very small and weakly fluorescent colonies.. after i pick the colony and innoculate them into new wells, the fluorescence just disappear.. maybe becoz the fluorescence are too weak..and the transfection is not stable
but cells transfected with mutant and wildtype form bright green fluorescent colonies..wonder what's the problem...
i think cells transfected with empty vector should have least impact on the cells...but it's not the case
Coolku
Yes, Cells seem dont like EGFP very much. I did on 293 cells and was able to select stable colonies but all of them have much weaker signals, at low to medium low levels when compared to transiently transfected. I dont know if your fusion protein altered the harmful effect of EGFP or not. What proteins are you working on?
-genehunter-1-
QUOTE (genehunter-1 @ Jun 6 2008, 12:30 PM)
QUOTE (coolku @ Jun 6 2008, 11:21 AM)
plasmid : PIRES2-EGFP
I transfected cells with PIRES2-EGFP carrying mutant gene, wildtype gene, or empty vector (but should still express EGFP), grow them in G418 for selection
i have repeated experiments several times..
each time：
cells transfected with the empty vector doesn't form any good fluorescent colonies...there are only very small and weakly fluorescent colonies.. after i pick the colony and innoculate them into new wells, the fluorescence just disappear.. maybe becoz the fluorescence are too weak..and the transfection is not stable
but cells transfected with mutant and wildtype form bright green fluorescent colonies..wonder what's the problem...
i think cells transfected with empty vector should have least impact on the cells...but it's not the case
I transfected cells with PIRES2-EGFP carrying mutant gene, wildtype gene, or empty vector (but should still express EGFP), grow them in G418 for selection
i have repeated experiments several times..
each time：
cells transfected with the empty vector doesn't form any good fluorescent colonies...there are only very small and weakly fluorescent colonies.. after i pick the colony and innoculate them into new wells, the fluorescence just disappear.. maybe becoz the fluorescence are too weak..and the transfection is not stable
but cells transfected with mutant and wildtype form bright green fluorescent colonies..wonder what's the problem...
i think cells transfected with empty vector should have least impact on the cells...but it's not the case
Coolku
Yes, Cells seem dont like EGFP very much. I did on 293 cells and was able to select stable colonies but all of them have much weaker signals, at low to medium low levels when compared to transiently transfected. I dont know if your fusion protein altered the harmful effect of EGFP or not. What proteins are you working on?
I just noticed that your constructs have IRES sequence, so no fusion proteins here, although the expression levels can be different with an insert. hmm. What about at the initial phase of the experiment? Did the cells transfected with the parent vector look dimmer/weak as well?
-genehunter-1-
QUOTE (genehunter-1 @ Jun 6 2008, 10:09 PM)
QUOTE (genehunter-1 @ Jun 6 2008, 12:30 PM)
QUOTE (coolku @ Jun 6 2008, 11:21 AM)
plasmid : PIRES2-EGFP
I transfected cells with PIRES2-EGFP carrying mutant gene, wildtype gene, or empty vector (but should still express EGFP), grow them in G418 for selection
i have repeated experiments several times..
each time：
cells transfected with the empty vector doesn't form any good fluorescent colonies...there are only very small and weakly fluorescent colonies.. after i pick the colony and innoculate them into new wells, the fluorescence just disappear.. maybe becoz the fluorescence are too weak..and the transfection is not stable
but cells transfected with mutant and wildtype form bright green fluorescent colonies..wonder what's the problem...
i think cells transfected with empty vector should have least impact on the cells...but it's not the case
I transfected cells with PIRES2-EGFP carrying mutant gene, wildtype gene, or empty vector (but should still express EGFP), grow them in G418 for selection
i have repeated experiments several times..
each time：
cells transfected with the empty vector doesn't form any good fluorescent colonies...there are only very small and weakly fluorescent colonies.. after i pick the colony and innoculate them into new wells, the fluorescence just disappear.. maybe becoz the fluorescence are too weak..and the transfection is not stable
but cells transfected with mutant and wildtype form bright green fluorescent colonies..wonder what's the problem...
i think cells transfected with empty vector should have least impact on the cells...but it's not the case
Coolku
Yes, Cells seem dont like EGFP very much. I did on 293 cells and was able to select stable colonies but all of them have much weaker signals, at low to medium low levels when compared to transiently transfected. I dont know if your fusion protein altered the harmful effect of EGFP or not. What proteins are you working on?
I just noticed that your constructs have IRES sequence, so no fusion proteins here, although the expression levels can be different with an insert. hmm. What about at the initial phase of the experiment? Did the cells transfected with the parent vector look dimmer/weak as well?
Thank you for your replay
After selection phase, when transfected cells started to express EGFP, the percentage of fluorescent cells (transfected with EMPTY VECTOR without inserting any gene) was quite low, and very weak...most were nonfluorescent, only G418 resistant..
I really thought cells transfected with mutant gene （histone gene）should have lower EGFP levels....
-coolku-