2.8 Site Directed Mutagenesis

ADAM10 and SOX2 reporter constructs with mutations in promoter or 3’UTR were generated by incorporating mutations into the miR-140 binding site on ADAM10 3’UTR and SOX2 3’UTR as well as in SOX2 binding site on ADAM10 promoter by PCR-based site directed mutagenesis using Pfu Turbo DNA polymerase (Quickchange II Site Directed Mutagenesis Kit; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol and were verified by sequencing. The primers used for generating mutations-in miR-140 binding site on ADAM10 promoter are: 5'ctgatcattggtaaacgccaaagatgagtaatttgc-3' and SDM ADAM10 3’UTR: 5'-gcaaattactcatctttggcgtttaccaatgatcag-3'. For generating mutations-in miR-140 binding site on SOX2 3’utr are: 5'-gctcctaccgtcctaatagaacttttaaaaagtttttcgtag-3' and 5'-ctttttaaaagttctattaggacggtaggagctttgcag-3' and for generating mutations in SOX2 binding site on ADAM10 promoter are: -5'-gatgtgcgtggattcgcgttaacgatgtgacagagag-3' and 5'ctctctgtcacatcgttaacgcgaatccacgcacatc-3'