Dear Prof Gegenheimer,
My name is Yair Pilpel, and I am a post-doctoral researcher in the
Max Planck Institute in Heidelberg, Germany.
I am sorry to disturb you, but I have seen your thread on T7 RNA
polymerase terminator sequence. I have been working with a T7-based
expression system in mammalian cells, and have taken the promoter,
followed by an IRES sequence and my protein, and then the T7
terminator sequence. Thus far it seems to be working, even rather
well. However, I have noticed that in my cloning I have made a
mistake and cloned instead of the ("classic") terminator sequence:
TAGCAtaaCCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
the following sequence:
TAGCAggcatgcCCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
Don't ask me how that happened, long story...
I've marked the differences between my and the classic sequence.
In any event, I am now unsure whether or not my expression would be
reduced by this change. Since I've already used this basic vector to
clone in a pretty large number of proteins, I am naturally averse to
reclone this sequence. However,it can (and will be done) if
necessary. Do you think that this terminator sequence should still
be working?
Another question that I have regards the exact site of termination.
I was thinking of using the T7 system to clone short-hairpin RNA's.
The thing here is that I would then need the termination point to be
very close to the beginning of the termination sequence. At least I
would need to know that there are no secondary hairpins being formed
after my RNA sequence. I guess probably that this is not the best
expression system for shRNA's, but would greatly appreciate your
opinion.
Thanks a lot,
Yair
