I am trying to find a time range of eGFP perdurance (on mouse embryo cells)- with no success. Does anybody know how long would the protein typically last once the gene it reports is not expressed?
Thanks a lot !!
Cheers
They begin to express at 30~48h post-transfection (hpt), and then gradually disappear at 72hpt. embryonic cells, specially the embryonic cells are quite proliferative. If you injected into early blastomeres, then it appears earlier than the transfection. If the embryonic cells are primary and highly proliferative, the transfection is not an easy task and can be improved greatly using commercial liposomal products. If you do the same in mammalian cell lines, the expression level is good to follow at transient expression. With G418 selection, you could establish a GFP cell lines, i.e., Histone 2B-GFP trasnfected cell line that give chromosomal behavior and other anticancer drug screening. You could see GFP expression within 18hpt earliest via electric chamber transfection in cell lines of cells (CHO, HeLa, MCF7, MDA-MB231, BV2, N2a, PC12, P19 EC and mES cells).
Hello Sang, thanks for your answer
I was rather looking for the time the GFP protein can last itself. For example, if I have a reporter mouse line expressing GFP under a certain promoter and then the promoter gets inactive, how long the GFP protein produced will usually perdure in the cell. Could it be days or weeks or is it just a matter of hours that it will get degrated?
Cheers
Sorry for that.
Gene insertion should be established to obtain the GFP fusion protein or the promoter-directed GFP expression for a prolonged time, perhaps permanently. Some transgene may be unstable, but works published shows that transgenes are sustained over cell cycles and even the generations of the transgenic animals. It does not matter whether the target-GFP protein or the promoter specific-GFP expression. There is no clear rule for how long. It depends on transient expression or expression through permanent insertion. If you are lucky enough to get a few cells with random gene insertion. The inserted gene survives over the replication and cell division, the promoter of interest will be turned on, showing a specific cell lineage of cells with GFP expression (probably many copies of your plasmid in tandem repeat within the mouse chromosomes). Fewer copies of gene insertion may be too weak GFP expression to trace the cell lineage. So successful insertion of many copies is a key for your purpose. The only drawback is you do not know whether the gene insertion and expression are successful until the time of cell differentiation. It could be overcome by cotransfection with CMV-RFP plasmid and the promoter-GFP plasmid together. Red fluorescence expression would tell transfection efficiency in most of the cells after transfection. By doing so, you can improve the efficiency by changing many conditions.
I recommend a high concentration of two gene constructs that will increase insertion of multiple copies of the promoter-GFP gene. Once inserted, some of the genes will be stable, and it will cover the duration of experiments just as in transgenic animals.