Retroviral delivery of siRNA directed against TEL-PDGFβR (T/P) breakpoint into hematopoietic cells. (A) The upper panel shows a schematic representation of TEL-PDGFβR fusion tyrosine kinase. The fusion junction that the siRNA was designed to target is indicated (upper and lower case letters correspond to TEL and PDGFβR sequence, respectively). The lower panel shows the predicted short hairpin transcript with a 3′_U₅ pol III stop signal. (B) Schematic representation of pQCXIP_ H1_TPsiRNA retroviral vector. The cassette of siRNA hairpin structure driven by pol III promoter H1 was subcloned into the 3′ ØLTR of the self-inactivating retroviral vector pQCXIP. Ø indicates deletion. Pcmv, CMV promoter; IRES, internal ribosomal entry site; Puro^r, puromycin-resistance; H1, pol III RNAse P RNA H1 promoter. (C) Expression of TEL-PDGFβR in stably transformed Ba/F3 cells. pQCXIP_H1_TPsiRNA was introduced into TEL-PDGFβR stable Ba/F3 cells following retroviral transduction. TEL-PDGFβR was detected with a rabbit polyclonal antibody recognizing PDGFβR C-terminal tail. Ba/F3 cells transduced with an empty retroviral vector, as well as stable Ba/F3 cells expressing TEL-PDGFβR transduced with pQXCIP retroviral vector encoding a nonspecific siRNA construct, were included as controls. WB, Western blot. (D) RT-PCR analysis with total RNA from different stable Ba/F3 cells was performed with a pair of primers amplifying approximately 350 bp sequence overlapping the breakpoint of TEL-PDGFβR. The mRNA level of TEL-PDGFβR was normalized to the endogenous GAPDH mRNA level as indicated. (E) Northern blot analysis with total RNA from different stable Ba/F3 cells was performed and probed with ³²P-labeled antisense 19-nt oligonucleotide for TPsiRNA. The control 5S-rRNA band was detected with ethidium bromide staining. Ba/F3 cells transduced with empty retroviral vector were included as a control.