4.9. Gene Knockdown by shRNAs
To knock down the expression of eEF1A in PK-15 cells, a lentivirus vector-mediated shRNA targeting eEF1A was constructed. Briefly, the upper and lower sequences of shRNAs for eEF1A and non-targeting negative control (NC) are listed in Table 1. The oligonucleotides were annealed and cloned into the lentivirus vector pLVX-shRNA2 (Clontech). The resulting recombinant plasmids pLVX-eEF1A-shRNA1, pLVX-eEF1A-shRNA2 or pLVX-NC-shRNA were cotransfected with the packaging plasmids psPAX2 and pMD2.G into HEK293T cells in 10-cm cell dishes. At 48 hpt, the lentiviruses in the culture supernatant were concentrated at 6000 ×g for 20 min at 4 °C using an Amicon Ultra-15 centrifugal filter unit with Ultracel-100 membrane (catalog no. UFC910096; Millipore, Billerica, MA, USA). The lentiviuses were titrated and transduced into PK-15 cells as described above. Knockdown of eEF1A was validated by Western blotting using the anti-eEF1A MAb (1:2000).