Specific and efficient Jak2^V617F gene silencing by the shRNA-encoded pRRL-mt4 lentivirus. (A) Sequences of the mt1- and mt4-shRNAs. The 9-nucleotide–long loop is indicated in bold, and the BglII and HindIII sites are underlined. (B) Schematic representation of the shRNA shuttle (pBS-H1) and lentivirus (pRRL) vectors. The shRNA was directionally inserted into the BglII and HindIII sites of the pBS-H1 vector. The expression cassette comprising the shRNA under the control of the H1 promoter was excised from the shuttle vector as a XhoI fragment and inserted into the lentiviral vector. LTR indicates long terminal repeat; cPPT, central polypurine tract; WPRE, posttranscriptional cis-acting regulatory element of the woodchuck hepatitis virus.^18,19  (C) JAK2 protein expression level in pRRL-mt1– or pRRL-mt4–infected HEL, UKE-1, SET-2, K562, and UT-7 cells. The cells were infected with the pRRL-Scr (control), pRRL-mt1, or pRRL-mt4 (both specific for Jak2^V617F) virus, as indicated. Transduced cells were selected for GFP expression, and protein contents were analyzed by Western blotting. HEL and UKE-1 cells only express JAK2^V617F, SET-2 expresses both JAK2^V617F and JAK2^WT, and K562 and UT-7 cells only express JAK2^WT. Inhibition of JAK2^V617F protein production in HEL and UKE-1 cells was observed after pRRL-mt1 infection and to a greater extent with pRRL-mt4 virus. Similarly, pRRL-mt4 infection decreased JAK2 content in SET-2 cells. As expected, pRRL-mt4 transduction had no effect on JAK2^WT protein production in K562 and UT-7 cells. (D) Jak2^V617F mRNA expression level, using allele-specific quantitative reverse transcription–PCR analysis, in UKE-1 and SET-2 cells transduced with pRRL-Scr or pRRL-mt4. Results represent ratios of Jak2^V617F over actin mRNAs, normalized to cells infected with pRRL-Scr. The differences in RNA and protein suppression efficiencies may be explained by the fact that data originated from 2 separated experiments or that JAK2 protein and mRNA are regulated independently.