Figure 8.
sh1005 can effectively reduce the levels of endogenous CCR5 mRNA in macrophages derived from FL-CD34+ cells. FL-CD34+ cells (2 × 105) were transduced with lentiviral vectors expressing sh1005 or Luc shRNA. The cells were harvested 3 days after virus transduction and sorted by the EGFP expression. The cells were then differentiated to macrophages with 20 ng/ml of IL-3, 50 ng/ml of SCF and 20 ng/ml of MCSF for 9 days and then with 5 ng/ml of GM-CSF for 5 days. Total RNA were isolated from macrophages using the Qiagen RNeasy extraction kit and treated with DNase I. Quantification of mRNA was performed using IQ5 with iScript one-step RT-PCR kit. RNA standards for CCR5 mRNA quantification were made by serial dilution of in vitro transcribed human CCR5 RNA using T7 RNA polymerase