Gene silencing mediated by lentivirus-encoded shRNAs in K562 cells. (A) The shRNA is transcribed from a human H1-RNA promoter inserted into the U3 region of the lentiviral Δ3′-LTR. The vector encodes RFP as a marker gene driven by the SFFV-LTR promoter and harbors a cPPT/CTS sequence. 5′-LTR indicates HIV-1 5′-LTR; Δ3′-LTR, HIV-1 self-inactivating (SIN) 3′-LTR; GA, deleted gag sequence; RRE, Rev responsive element; SD, splice donor site; SA, splice acceptor site; Ψ, packaging signal, SFFV-LTR of spleen focus-forming virus. (B) SHP2, STAT5, and Gab2 mRNA levels were measured by real-time RT-PCR 4 days after lentiviral transduction and normalized in comparison to GAPDH expression. The shRNAs are indicated on top of each bar. mRNA expression of SHP2 (left), STAT5 (middle), and Gab2 (right) in control dcH1-GL4-SR-transduced cells was set to 100% (average of 3 independent experiments). (C) For immunoblotting, cells were transduced with control dcH1-GL4-SR (lane 2), dcH1-b3a2_1-SR (lane 3), anti-SHP2 shRNAs 237, 594, and 714 (top: lanes 4-6), anti-STAT5 shRNA (middle, lane 4), and anti-Gab2 shRNA (bottom, lane 4) and lysed 6 days after transduction, respectively. Lane 1 shows untransduced K562 cells. The immunoblots were probed with anti-SHP2, anti-STAT5, and anti-Gab2 antibodies, respectively, and reprobed with anti-ERK2 antibody as loading control. (D) Number of K562 cells negative for trypan blue after lentiviral transduction with different shRNAs is shown (averages from 3 experiments). One milliliter with 104/mL cells was plated, cultures were split and fed twice a week, and calculated cell numbers are indicated. Triangles depict transduction with dcH1-GL4-SR (control), and circles show transduction with viruses dcH1-b3a2_1-SR, dcH1-237SHP2-SR, dcH1-Gab2-SR, and dcH1-STAT5-SR, respectively.
