Team:UT-Tokyo/CTCD/Content/Result

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Our aim was to make a construct like below.(fig.1)

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miRNA142-5p sequence is CAUAAAGUAGAAAGCACUACU .
miRNA142-3p sequence is UGUAGUGUUUCCUACUUUAUGGA.

The target sequence is complementary to the miRNA142-5p and miRNA142-3p sequence.

miRNA142-5p binding site is AGTAGTGCTTTCTACTTTATG.
miRNA142-3p binding site is TCCATAAAGTAGGAAACACTACA.

We constructed miRNA-binding sites by annealing the two oligos shown below.

5’-GCGGAATTCGCGGCCGCTTCTAGAGCATAAAGTAGAAAGCACTACTTACTAGTAGCGGCCGCTGCAGGCG-3’
5’-CGCCTGCAGCGGCCGCTACTAGTAAGTAGTGCTTTCTACTTTATGCTCTAGAAGCGGCCGCGAATTCCGC-3’

When we made miRNA-binding site connected tandemly, we couldn’t use PCR or gel extraction because they contained repeats and short sequences . So we had to anneal two oligos and insert in the plasmid.(fig.2)

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This method can be used for other miRNA-binding sites or other parts not suitable for PCR or gel extraction .

Constructs we planed to use in this experiment were already made apart from EGP-2 promoter, and sequences were confirmed. But cloning of EGP-2 was not successful.
So we have to clone EGP-2 promoter, make the construct,and perform reporter assay before our presentation.