Cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector.
Related Journal & Article Information
Journal: Nature Cell Biology
Article Title: The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation
Introduction
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).
Materials
Reagents
Equipment
Procedure
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.
2. «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.
3. «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.
Troubleshooting
Critical Steps
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.
Anticipated Results
References
1. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell. 2003 Dec 26; 115(7): 787-98.
Acknowledgements
We thank Dr. D. Bartel for kind gift of pISO vector
Keywords
sceletal muscle, miRNA target, luciferase vector
Figure 1
Cloning of amplified target miRNA sites into 3’UTR of luciferase reporter vector.
