general question- how to study regulation of gene expression - (Nov/21/2005 )

Hey guys, just a general question here
If u have a gene of unknown sequence, how would you study the regulation of gene expression of it?
And if it's of known sequence, what difference would be included the study approach?
thanks very much

Hmmm...if its of unknown sequence I can't think of any way its regulation could be monitored. You would need the sequence to run a northern blot which allows you to quantify the mRNA of the gene under different conditions. For example, you could grow cells under different temperature, extract RNA and run a northern blot to compare the abundance of the specific gene mRNA under each treatment. Alternatively, you could fuse the promoter of the gene to a marker gene like lacZ or gfp and measure the end product of the marker genes (ie. B-galactosidase activity or fluorescence respectively) as it is exposed to different treatments (ie. heat or other stress).
How do you know the gene exists in the cell you are looking at if you haven't got the sequence? Did you amplify it from the genome?

1) Use degenerate primers obtained from similar organisms to amplify it out (search literature for this)

2) Sequence the PCR product.

3) Create specific primers within the sequenced product that amplify for 300-500 bp.

4) Use qRT-PCR and a relative, constituitively active standard to quantify the cDNA/RNA.

-Matt