High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

For construction of the human Dicer expression vector pTT5-Dicer, a PCR fragment encoding Dicer was first generated from plasmid pFRT/TO/FLAG/HA-DEST DICER [provided as a gift from Thomas Tuschl (Addgene plasmid # 19881)] [48] and inserted between the NotI and HindIII sites of the pTT5SH8Q1 vector to allow for expression of Dicer with a C-terminal StrepTagII/His 8 tag [34] . The expression vector for the catalytically-inactive Dicer D1320A/D1709A variant was prepared from pTT5-Dicer using the Stratagene QuikChangeII site-directed mutagenesis method. Plasmids were amplified in Escherichia coli (E. coli) DH5α cells (Invitrogen) grown in LB medium supplemented with 100 mg/L amplicilin and purified using the QIAGEN Plasmid Giga Kit. Plasmids were stored at 4°C in water at a concentration of 1 mg/mL.