Inserting a reporter into the genome - (Feb/15/2010 )
Hi all. I want to insert a GFP reporter behind the promoter of a protein of interest such that if that certain gene is expressed then GFP will be translated as well. I want to do this in a primary human fibroblast cell line. I don't know much about manipulating mammalian genomes, but how difficult would it be to do what I'm describing? Do you know the time scale and how much troubleshooting/headache it would be? I've been trying to find protocols for this, but I think I'm using the wrong search terms because I keep finding protocols for generating transgenic mice. Thanks!
-scientistI-
scientistI on Feb 15 2010, 02:42 PM said:
Hi all. I want to insert a GFP reporter behind the promoter of a protein of interest such that if that certain gene is expressed then GFP will be translated as well. I want to do this in a primary human fibroblast cell line. I don't know much about manipulating mammalian genomes, but how difficult would it be to do what I'm describing? Do you know the time scale and how much troubleshooting/headache it would be? I've been trying to find protocols for this, but I think I'm using the wrong search terms because I keep finding protocols for generating transgenic mice. Thanks!
This is possible, but very difficult and very unlikely to be worth the effort. It would require achieving genomic integration at a precise location, rather than just general integration as is achieved through most stable transfection techniques. Why not just look for gene expression via qPCR or in situ hybridization? Has the promoter been characterized?
-Dr Teeth-