Gene targeting - (Nov/29/2010 )
Hi, I need a little help on this one. If I just isolate a new gene that I think could have target the NF-kB pathway. What experiment I can perform to show that the new gene target NF-kB pathway?
Thanks
That depends on how it may target the NFkB pathway...
You may want to overexpress and/or knock down your gene and check levels of NFkB genes by qPCR, or perform luciferase assay with the NFkB promoter and your GOI, or check levels of NFkB with western, NFkB phosphorylation, degradation, NFkB target gene stimulation or repression, gene networks by microarray... You can also do a mouse model, knock it out, overexpress, and hope that you get a phenotype.
Lots of things to do!
rsm
Rsm on Tue Nov 30 07:43:41 2010 said:
That depends on how it may target the NFkB pathway...
You may want to overexpress and/or knock down your gene and check levels of NFkB genes by qPCR, or perform luciferase assay with the NFkB promoter and your GOI, or check levels of NFkB with western, NFkB phosphorylation, degradation, NFkB target gene stimulation or repression, gene networks by microarray... You can also do a mouse model, knock it out, overexpress, and hope that you get a phenotype.
Lots of things to do!
rsm
If I decide to perform a luciferase assay, how should I make my vector construct? I am sorry I am new to this field.
Ususally you take 1 or 2kb of a specific NFkB promoter, and replace the gene with luciferase. Then you co-infect cells with this plasmid and an expression plasmid encoding your gene of interest, and measure luciferase activity after 48hrs. You need to include some controls, like reporter plasmid only etc... But I'm not a specialist in this, maybe you'll find someone in your department who can help you?
rsm