Express miRNA in vector for gene knockdown - (Sep/22/2006 )

Hello,

I am trying to clone miRNA under a polII promoter into a lentiviral vector for knockdown of a specific gene. Do I need to have a polyA tail immediately downstream of the miRNA to terminate the transcription unit.

Thanks in advance,
Anita.

Read thru this article : Design and cloning of lentiviral vectors expressing small interfering RNAs, Gustavo Tiscornia, Oded Singer and Inder M Verma.

I dont know if u need a polyA for pol II but for pol III u dont need.

Hi!
I think you should never clone a poly A into the "open reading frame" of a lentivirus vector (i mean between the LTRs) because then you get early termination of your mRNA and it can not be packaged properly...please correct me if i'm wrong...

Stardust

Hi,
Thanks so much for your replies. You are right, the 3'LTR already has a poly A tail, thus if I insert another one it will lead to premature termination of the transcript.
This also makes my cloning process more feasible. Thank you!
Anita.