why EGFP become weak in C2C12? - (Nov/26/2006 )
hi, I have some trouble with EGFP expression in mouse-derived cell.
here is what troubles me:
C2C12 was infected with lentivirus system, and then the positive cells (EGFP expression cells) were selected by FACS, first, all the cells is green, after several passages, the EGFP become weak, and a lot of cells showed no EGFP expression, why?
I have been told that EGFP could be lost even in a stable cell line, but I don't know why. can anyone give me information about this?
Any suggestions are welcome, thanks in advance.
here is what troubles me:
C2C12 was infected with lentivirus system, and then the positive cells (EGFP expression cells) were selected by FACS, at first, all the cells is green, after several passages, the EGFP become more and more faint, and a lot of cells showed no EGFP expression, why?
I have been told that EGFP could be lost even in a stable cell line, but I don't know why. can anyone give me information about this?
Any suggestions are welcome, thanks in advance.
If you are not selecting your cells, it is normal you lose EGFP because the vector doesn't have an eukariotic origin of replication: when cells divide, the plasmid will go only to one of the two daughter cells, unless genomic integration has occurred.
Regarding EGFP becoming more and more faint, if you imagine several copies of the plasmid in one cell, the more the cell divides, the fewer copies of the plasmid daughter cells get (same reasons as above), the less RNA is transcribed and protein translated.
Hope this makes things a bit more clear
Regarding EGFP becoming more and more faint, if you imagine several copies of the plasmid in one cell, the more the cell divides, the fewer copies of the plasmid daughter cells get (same reasons as above), the less RNA is transcribed and protein translated.
Hope this makes things a bit more clear
thank you,but what I used is lentivirus system, which can integrate into chromosome.
when hela cell was infected by this system, the EGFP was stable even several passages,
but in C2C12, it seems not so good.......
In murine retroviruses vector system, it has been known for some silencing effect on the LTR region, especially in primitive cells. there are some protein binds to the U3 region and block the transcription followed by methylation of the LTR. There are some reports showing that such silencing effect also happen in lentivirus vector. I'm not sure about the C2C12 cells, but from what you had been described, it seems like it's a suppression effect. check out those retrovirus and lentivirus vectors papers on how they develops those vectors. you might have some clues. Good Luck and happy new year!
Haemophilic