GFP GOLGI - Expresion of pAcGFP Golgi. (Jan/30/2007 )
Hello¡
I am trying to make estable lines of RPE-1 cells that express pAcGFP GOLGi (http://www.clontech.com/products/detail.asp?tabno=2&catalog_id=632464&page=all) but i am having a lot of problems, because the clones isolated derive and only have resistence to the antibiotic but no show the fluorescent Golgi.
Can everybody help me?
Sorry me for my English.
Thanks.
We have seen many times that transfected cells in selection media grow but donot have the transfected gene. I dont know the reason.
We normally screen 6 clones and get 1-2 positive clones although all of them have been in the selection antibiotics for some time.
Best suggestion would be to screen more clones.
It could help to increase antibiotic conc. for selection.
I am trying to make estable lines of RPE-1 cells that express pAcGFP GOLGi (http://www.clontech.com/products/detail.asp?tabno=2&catalog_id=632464&page=all) but i am having a lot of problems, because the clones isolated derive and only have resistence to the antibiotic but no show the fluorescent Golgi.
Can everybody help me?
Sorry me for my English.
Thanks.
your stable cell lines may carry both genes (antibiotics resistant factor and GFP GOLGI marker) but GFP-fused protein may not shine bright; check green fluorescent with a very sensitive CCD; I think degree of expression depends, among other things, on how many copies of your genes are integrated in the genome; so as suggested select and qualify as many clones as you can to get some properly expressing cell lines
I think that isn´t the problem, my cells at the first time have golgi fluorescense, but along the time the cells lost GFP in a progresive maner, but are resistent to antibiotic.