I am trying to obtain a stable transfection of a plasmid containing a gene of interest. The plasmid I am using is from invitrogen and it is called pTracer-EF/BSD. The plasmid contains a blasticidin resistance gene that gives rise to a GFP-blasticidin resitance fusion protein. At first I was trying to use lipofectamine 2000 to generate a stable transfection. But, after about 7 days I cannot detect any flourscence any more. Even on colonies surviving the blasticidin treatment. I also tried a protocol for electroporation (protocol claims to be optimized for production of stable cell lines). Using electroportaion, I did not see any GFP flourescence at all; not even after 3 days!
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?
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#2
expresson_help
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Posted 10 February 2005 - 06:57 AM
I don't think only having one copy of the gene inserted will be the problem, as assuming that it's under a strong promoter, thousands of copies of RNA will result from it.
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.
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.
#3
chicho
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Posted 10 February 2005 - 10:27 AM
In all the transfections and electroporations I have used linear DNA. If it is true that only one copy of the gene is enough for flourescence, then lipofectamine 2000 should be able to give me some stable transfectants.
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?
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?
