Cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector.

doi:10.1038/nprot.2006.156

Method Article

Cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector.

https://doi.org/10.1038/nprot.2006.156

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Biotechnology

Biochemistry

Molecular Biology

sceletal muscle

miRNA target

luciferase vector

Four copies of miR-181a target site from mouse HoxA-11gene have been cloned between SacI and XbaI sites of pISO vector (kind gift of David Bartel), containing a MCS downstream of Firefly Luciferase ORF.

The cloning strategy used compatible cohesive ends SpeI/XbaI to enter sequentially 1,2,4 etc. copies of target sequences (Figure 1).

The oligonucleotide duplex containing predicted 43 nt Hox-A11 target sequence for miR-181a, 5’-Sac I, Spe I, 3’-XbaI restriction sites and 5’ Sac I and 3’Xba I sticky ends was cloned into Sac I/Xba I sites of pISO vector. This construct « 1Target plasmid », was sequenced and used for further cloning. Notice that Sac I and Xba I sites in extremities of fragment have been destroyed after ligation.
«1 Target plasmid» was either linearized by SacI/SpeI restriction or cut by SacI/XbaI to obtain the fragment containing 1 target. The fragment was gel-purified and ligated into linearized plasmid. Resulting construct containing duplicated target site, « 2 Targets plasmid », has been used for further target amplification.
«2 Targets plasmid » was linearized by SacI/SpeI restriction. SacI/XbaI fragment, containing 2 targets was cut, gel-purified and ligated into linearized « 2 Targets plasmid ». Resulting construct contained 4 target sites.

Before cloning, check miRNA target sequence for the presence of eventual restriction sites using for cloning. If it is the case, use another compatible cohesive ends.

Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell. 2003 Dec 26; 115(7): 787-98.

We thank Dr. D. Bartel for kind gift of pISO vector

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Cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector.

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Cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector.

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