Lentivial Knockdown of miRNAs - (Jan/07/2010 )

Is there anyone here doing lentiviral knockdown of miRNAs? Any vectors that you would recommend? I can only find a vector called "miRZip" from system bioscience which is for this purpose.

Also, do you know what should be cloned into the miRZip vector? Thanks.

Samantha

Two options are to make an shRNA targeting the loop section of the pre-miRNA molecule, or clone in a synthetic miRNA target for the miRNA to bind to (i.e. like a microRNA sponge). I'm sure that there are other possibilities though...

miRNA man on Jan 7 2010, 11:36 AM said:

THanks for your reply, miRNA man. Could you explain a bit more on that, or are there any references/links that I can take a look?

Samantha

Here are references for the sponge: 17694064 and 18931683 (PubMed ID numbers), and 15722555 for the shRNA against the hairpin loop (they used chemically modified siRNA not shRNA, but I think it could still work well).

hi!I am from China.I am interested in knockdown miRNA by Sponges.Anyone have use it in research?how to construct the plasmid?thanks!

Your can read the original paper from Philp Sharp lab. You need to clone multiple copies of the miRNA target into the 3UTR of a gene, for example, luciferase.

pcrman on Apr 2 2010, 10:43 AM said:

Thank you,pcrman!I am constructing a spnoges for a miRNA , only promoter + sponges without luciferase, and hope it could work well!

Why don't you want to include a reporter gene like luc as pcrman suggested? It should be useful to track expression and besides, I personally doubt if a sponge would work without a gene (the mRNA needs to be exported to the cytoplasm to be functional...will this happen if there is no CDS?)

miRNA man on Apr 3 2010, 05:48 AM said:

Thank you! At first, I think I could detect the amount of mature miRNA to test the sponges work, Now I will add a report gene on upstream of Sponges.
To miR ,I am a new. Thank you!