How do I terminate transcription at a specific site in a plasmid that transfected into mammalian cells???

michael.baron at bbsrc.ac.uk michael.baron at bbsrc.ac.uk
Mon Aug 12 09:14:16 EST 1996


You say you want to produce the RNA in the cytoplasm. Easiest way to do this is to 
use a T7 RNA pol promoter vector and a recombinant vaccinia expressing T7 RNA 
polymerase. The new recombinants based on the host-range restricted MVA strain are 
very good (no CPE, good T7 expression, no vaccinia). Down side is that you get your 
RNA capped, which may or may not be a problem.

To get *exact* termination at the end of your insert you should have a hep delta 
virus ribzyme sequence just downstream of your insert. Your transcript will then 
cleave off everything you don't want by itself!
There are a number of publications where people have used such constructs. I'd send 
you mine but, this being an animal virus research lab, some countries refuse to have 
anything posted from here in the country!

regards,

Michael




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