8 replies to this topic

[Samantha]

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

[miRNA man]

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...

[Samantha]

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

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...

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

[miRNA man]

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).

[redhorse]

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!

[pcrman]

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.

[redhorse]

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

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.

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

[miRNA man]

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?)

[redhorse]

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

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?)

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!