6 replies to this topic

[altobarn]

Hi all,

I've got a bunch of miRNAs which predicted to be targeting some candidate target genes and I've been working on Luciferase assays to comfirm their real functional effect. I have selected some interesting miRNAs to proceed to the next step, which by blocking the binding site (seed sequence) of the miRNA to mRNA. Anyone has any experience how to make it (site mutagenesis) or where to buy it (target protector/blockmir)?

My another problem for site mutagenesis is to which nucleotide of the seed sequence should I mutate to? Also, what nucleotide should I replace so that the miRNA that I transfect wouldn't be able to bind to mutagenated site? Any software or 'rules of thumb' for this?

Any comments, suggestions would be very much appreciated. Many thanks!

-altobarn-

Edited by altobarn, 14 July 2009 - 01:25 AM.

[Jon Moulton]

altobarn, on Jul 14 2009, 02:24 AM, said:

...I have selected some interesting miRNAs to proceed to the next step, which by blocking the binding site (seed sequence) of the miRNA to mRNA. Anyone has any experience how to make it (site mutagenesis) or where to buy it (target protector/blockmir)? ...

Morpholino oligos are good target protectors.

Choi WY, Giraldez AJ, Schier AF. Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430. Science. 2007 Oct 12;318(5848):271-4. Epub 2007 Aug 30.

Lin Z, Murtaza I, Wang K, Jiao J, Gao J, Li PF. miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy. Proc Natl Acad Sci U S A. 2009 Jul 2. [Epub ahead of print]

Gene Tools makes them. www.gene-tools.com I work for Gene Tools.

Edited by Jon Moulton, 14 July 2009 - 10:19 AM.

[altobarn]

Many thanks Jon! Actually I've been surfing gene-tools website too. In order to get the target proctector, do I just need to tell gene-tools which 3'UTR mRNA need to be protected from a certain microRNA, and then the company will synthesize for me?

Concerning site mutagenesis, does anyone else has any clue?

Many thanks

-Altobarn-

[Functional Screens]

We have used 500 human miRNA lentiviruses (~100 bp flanking sequences on either side of pre-miRNA cloned into a lentiviral expression vector to generate lentivirus) to screen miRNA which inhibits breast cancer metastasis. We got a few positive hits and to further confirm, I have changed the first 10 nt on the mature miRNA candidate sequences to GAGAGAGAGA and flip the inserted sequence (~300bp) as well to serve as controls. The data have not been published yet, however this is what we did. Alternatively, You can mutate or delete your target sequences (on the 3'UTR) to show no suppression while you change them. Hopefully this helps.

[Jon Moulton]

altobarn, on Jul 15 2009, 04:45 AM, said:

In order to get the target proctector, do I just need to tell gene-tools which 3'UTR mRNA need to be protected from a certain microRNA, and then the company will synthesize for me?

Hi Altobarn,

Ideally you will identify the site you think the miRNA binds (the MRE). Send us about 50 bases of UTR sequence with the MRE in the middle. If the sequence is upper case, write the bases complementary to the miRNA seed sequence in lower case. We'll look for the best oligo target that covers the seed sequence binding site. You can email directly to me, jmoulton <at> gene-tools.com

[altobarn]

Thanks for the replies!

To Functional Screens - I'm not completely comprehended your explanation. But as far as I could see, it is possible to construct the site mutagenesis by myself and not necessary to buy from company? Is there any links or reference teaching us how to make it?

To Jon - Thanks again. I will email you shortly after.

[Functional Screens]

altobarn, on Jul 17 2009, 12:50 AM, said:

Thanks for the replies!

To Functional Screens - I'm not completely comprehended your explanation. But as far as I could see, it is possible to construct the site mutagenesis by myself and not necessary to buy from company? Is there any links or reference teaching us how to make it?

To Jon - Thanks again. I will email you shortly after.

If you already have the miRNA expression clones, you can start mutagenesis from there. Since you said you have carried out luciferase reporter assay, I assume you already have them. Otherwise, you might want to buy or make your own (clone 100-250 flanking sequences on both sides of pre-miRNA and cloned into an expression vector). Regarding how to choose a miRNA expression vector, I have made comments in the following link: http://www.protocol-online.org/forums/inde...?showtopic=9039

What I did was to change the first 10 nt sequence (to GAGAGAGAGA) on the mature miRNA of interest (within the 100bp flanking + pre-miRNA sequence in the expression clone) to generate my mutants. The data have not been published yet.

For your reference, you can either mutate mature miRNA sequence or target sequence on the 3'UTR of mRNA to validate its function.
Cell 2006 124 1169 A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors.
Nat Cell Biol. 2008 10 202 The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis.
Genes Dev. 2009 23 862 MicroRNA-125b is a novel negative regulator of p53.