erosenbe at gpu3.srv.ualberta.ca
Fri Nov 22 00:23:05 EST 1996
Paul N Hengen (pnh at ncifcrf.gov) wrote:
: Elizabeth Rosenberg (erosenbe at gpu2.srv.ualberta.ca) wrote:
: > Now we would like to insert an antisense fragment of the same gene into
: ---------------------------------^^^^^^^^^ ^^^^^^^^
: > the pREP10 vector and tranfect this construct....
: Oh Nooo. It's now being tranferred from ssDNA to dsDNA. An a-fragment???
: This thing spreads like a hungry virus. Run for your lives... :-o
: Whatever you do, stay away from the diner, and carry a fire extinguisher.
: [The Blob, Steve McQueen circa 1952]
OK. I said we were new to this game. I apparently don't have the jargon
down quite yet. Let's try this explanation: the pREP10 vector is
essentially the same as the pREP9 vector with the exception that the
promoter is situated such that we can take the same dsDNA sequence that
we used in the pREP9 vector to get "normal" gene expression, insert it
into the pREP10 vector and get transcription in the opposite orientation
resulting in an "antisense RNA fragment". From what I can gather, it is
unadvisable to use the full-length cDNA if you wish to target endogenous
mRNA for destruction. Therefore, we "plan" to use various restriction
fragments of our cDNA in the pREP10 construct. Hope this is more clear.
Is this an acceptable strategy?
: you probably already know by now that the "A" word WRT DNA is taboo :-)
Paul, thanks for reminding me what a mine field of lingo I've walked into.
Truly, thanks for the help, and I hope to learn much more. Is the above
closer to correct?
Beth Rosenberg erosenbe at gpu.srv.ualberta.ca
Cross Cancer Institute
University of Alberta "The sheep and the wolf do not
agree on the definition of liberty"
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