stable transfection of mouse embryonic stem cells
Posted 09 February 2005 - 03:23 PM
I was told by a fellow researcher that it will be hard to see the GFP flourescene at all if a cell has only one copy of the gene inserted. But is one copy of the gene enough to provide for blasticidin resistance? Would the FACS be able to detect the flourescence of such cells even if I cannot detect it by myself throught the microscope?
Anyone have any similar experiences, thoughts or ideas?
Posted 10 February 2005 - 06:57 AM
It osunds like your plasmid is not stably inserting into the genome. The probability of this occuring can be increased by linearising the plasmid prior to transfection with a restriction enzyme that doesn't disurb any functionally important element of the vector.
In addition, since you're using a primary cell line, your transfection efficiency might be very low, decreasing the probability of successful integration. You might want to consider using neophectin (http://www.expresson...neophectin.html) which is very good for transfection into primary cells.
Posted 10 February 2005 - 10:27 AM
I heard reports that the CMV promoter (the same promoter used for my blasticidin_res-GFP protein) is not very active in mouse embryonic stem cells. Maybe this is the cause of my inability to detect flourescence under the microscope. In this case, will the FACS be sensitive enough to detect it?
Anyone here with this kind experience?