Pol III Promoter Based miRNA Expression Vectors

The plasmid pPUR-U6 was constructed, which is originally from the pPUR vector (Clontech). A U6 cassette containing the human U6 promoter followed by two BbsI cloning sites and four strings of tyramine residues was inserted into the EcoRI and BamHI site of the pPUR vector. pol III promoter-based miRNA expression vectors constructed on pPUR-U6 should follow a procedure similar to that used in the pol II promoter-based miRNA expression vectors. RNA pol III terminates transcription at more than four T tracts; therefore, be sure that there are no T strings of more than 4 nt in the desired sequences of the miRNA precursor.

1. Amplify the template genomic DNA with specific primers of each miRNA by PCR reaction. Our PCR primers were designed to amplify the predicted miRNA precursor sequence with 30-bp flanking regions and BbsI sites in the 5' and 3'ends.

2. After purification of the PCR products by a PCR purification kit, digest 1 ^g of the vector plasmid pPUR-U6 and the PCR products with BbsI in NE buffer 2 for 2 h at 37°C in a total volume of 20 ^L.

3. After purification and ligation of the vector and the insert fragments, transform E. coli cells with the ligation mixture in the same way as set out in steps 7 to 11 in Subheading 3.1.2.

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