In this case, downregulation of Drosha using the siRNA technique should increase titers of miRNA-encoding retroviral particles. To test this hypothesis, we first determined the amount of Drosha-specific siRNA required to efficiently downregulate the enzyme by transfecting Phoenix cells via the calcium phosphate method with synthetic siRNA against Drosha. In western blots, the signal for Drosha was already reduced with 50 pmol and barely detectable with 800 pmol siRNA (Supporting Information Fig. 2). Next, we co-transfected Phoenix cells with a retroviral expression vector encoding miR-106b and selleck chemicals llc with
200 pmol of Drosha siRNA or a control siRNA against luciferase. As expected, western blot analysis verified the successful downregulation of Drosha only in cultures that were co-transfected with siRNA against Drosha (Fig. 1B). As revealed by flow cytometry, frequencies
of GFP-positive cells were quite GPCR Compound Library cost similar in all transfected Phoenix cultures, ranging from 87 to 98% (Fig. 1C). Downregulation of Drosha did not lead to altered abundance of Dicer, the second RNaseIII enzyme needed to release mature miRNAs from the hairpin precursors. siRNA-mediated downregulation of Drosha in Phoenix cells should increase the amount of viral particles in the culture medium of cells transfected with retroviral constructs. As summarized in Supporting Information Fig. 3, this was indeed the case. More importantly, flow cytometry detected approximately Fossariinae 80% GFP-positive
cells in NIH3T3 cultures that were infected with retroviral supernatants of Phoenix cells co-transfected with pCLEP-106b and 200 pmol Drosha siRNA (Fig. 1C), similar to the frequency of GFP-positive cells in NIH3T3 cultures infected with the empty control virus. In contrast, only 32 and 47% of NIH3T3 cells could be infected with miR-106b virus from Phoenix cells transfected without Drosha siRNAs or a control siRNA against luciferase, respectively. Transfection of Phoenix cells with 800 pmol of Drosha siRNA yielded a very similar picture (data not shown). We next confirmed the effect of Drosha siRNA with pCLEP-30c. Addition of siRNA against Drosha in the Phoenix transfection cocktail led to a three- to four-fold increase in GFP-positive cells in infected NIH3T3 cultures (data not shown). Therefore, titers of miRNA-encoding retroviral particles were increased by co-transfecting the packaging line with the retroviral expression vector and an siRNA against Drosha. To test whether the addition of Drosha siRNA also improves the transduction efficiency in primary B-cell cultures, we infected pre-activated primary splenic (CD43−) B cells with supernatants from Phoenix cells transfected either with the control vector pCLEP, with pCLEP-106b or with pCLEP-106b together with 200 pmol of Drosha siRNA (Fig. 1D).