Successful RPS19 mRNA knockdown by lentiviral vectors LV-TH-RPS19-A, -B, and -C. (A) The lentiviral construct contains an internal human EF1a promoter expressing enhanced GFP. The 3′ longer terminal repeat (LTR) that is duplicated in the integrated provirus contains a polymerase III-dependent H1 promoter that expresses an RNA hairpin, which is cleaved by the RNA-induced silencing complex to a 21nt siRNA molecule. The LTR also contains a TetO sequence capable of binding a transcriptional repressor, a feature that is not used in this study. cPPT indicates central polypurine tract; EF-1a, elongation factor-1a promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; and SIN, self-inactivating. (B) Northern blot analysis of total RNA from transduced CD34⁺ CB cells that were sorted for GFP shows increasing RPS19 silencing in vectors LV-TH-RPS19-A, -B, and -C. (C) Q-RT-PCR (mean from 2 independent experiments) confirms that vector LV-TH-RPS19-A is the least effective and vector LV-TH-RPS19-C is the most effective in the ability to silence RPS19 mRNA. (D) Representative Western blot of CD34⁺ CB cells sorted for GFP 5 days after transduction. (E) Densitometry analysis of Western blots (mean of 2 independent experiments) 5 days after transduction shows decreasing RPS19 protein levels in CD34⁺ CB cells transduced with LV-RTH-RPS19-A through -C.