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214 Zhang et al.Fig. 2. Generation of U6-shRNA-expression cassette by PCR. The U6-shRNAexpressioncassette is generated by PCR using plasmid pGEM-U6 as a template; a forwardprimer that contains a XhoI site and anneals to an SP6 site in the plasmid; a reverseprimer that contains an EcoRI site, the shRNA sequence, and anneals to the 3′-end ofthe U6 promoter. The PCR product is digested with XhoI and EcoRI and subcloned intopPUR V2 cut with the same two enzymes.18. Sterile-filtered, 1 mg/mL puromycin in phosphate-buffered saline, pH 7.3 (Clontech).19. U2OS cells grown in Dulbecco’s Modified Eagles (DME) supplemented with 10%(v/v) fetal bovine serum (FBS) in a humidified 37°C, 10% (v/v) CO 2incubator.20. Luria Bertani media + 100 µg/mL ampicilin (Sigma).21. Phoenix cells were a gift by Gary Nolan, and WI38 primary human fibroblast cellswere purchased from ATCC: Manassas, VA. Phoenix cells should be grown inDME supplemented with 10% (v/v) FBS in a humidified 37°C, 5% (v/v) CO 2incubator. WI38 cells should be grown in Dulbecco’s modified Eagle’s mediumsupplemented with 20% (v/v) FBS, essential and nonessential amino acids, andvitamins (Cellgro: Herndon, VA) in a humidified 37°C, 5% (v/v) CO 2incubator.22. 8 mg/mL Polybrene in ddH 2O (Sigma).23. 0.45-µm Filter (Fisher: Pittsburgh, PA).24. ddH 2O.3. Methods3.1. Generation and Validation of Functional shRNAs in pPUR V2The shRNAs are generated by PCR, as fusions to the human U6 RNApolymerase III promoter (Fig. 2). It is recommended to first construct 3–6shRNAs per gene and identify those that efficiently knockdown the target in a