Gene specific sirna oligonucleotides

Primary samples of CD34+ HSPCs were harvested by leukapheresis from healthy volunteers. The cells were purified to ≥98% CD34+ content by magnetic cell sorting using Clini MACS (Miltenyi Biotech, Bergisch-Gladbach, Germany) and cryopreserved in liquid nitrogen. Approval for this study was obtained from the Ethical Committee of Hannover Medical School, and informed consent was obtained in written form in accordance with the Declaration of Helsinki. T-ALL cell lines are held by the DSMZ (Braunschweig, Germany) and were cultivated as described elsewhere [26 ]. Gene specific siRNA oligonucleotides and AllStars negative Control siRNA (siControl) were obtained from Qiagen (Hilden, Germany). Expression constructs for HHEX, HLX1, NKX2-3 and NKX3-1 were cloned in vector pCMV6-XL4 and obtained from Origene (Wiesbaden, Germany). SiRNAs (80 pmol) and expression constructs/vector controls (2 μg) were transfected into 1x10⁶ cells by electroporation using the EPI-2500 impulse generator (Fischer, Heidelberg, Germany) at 350 V for 10 ms. Electroporated cells were harvested after 20 h cultivation.

T-ALL cell lines are held by the DSMZ (Braunschweig, Germany) and were cultivated as described previously [64 ]. PER-117 cells were kindly provided by Dr. Ursula Kees (Perth, Australia) [65 (link)]. Treatments of cell lines were performed for 20 h with 5 μM 3-Deazaneplanocin A hydrochloride (DZNep) (Sigma, Taufkirchen, Germany), 10 μg/ml Trichostatin A (TSA) (Sigma), indicated concentrations of ICBP112 (Sigma), or 20 ng/ml recombinant human Interleukin-7 (IL7) protein (R&D Systems, Minneapolis, MN, USA). Gene specific siRNA oligonucleotides and AllStars negative Control siRNA (siControl) were obtained from Qiagen. Expression constructs for AUTS2, PCGF5 and RUNX1 were obtained from Origene (Wiesbaden, Germany). SiRNAs (80 pmol) and expression constructs/vector controls (2 μg) were transfected into 1×10⁶ cells by electroporation using the EPI-2500 impulse generator (Fischer, Heidelberg, Germany) at 350 V for 10 ms. Electroporated cells were harvested after 20 h of cultivation. Lentiviral transduction of the STAT5 gene into JURKAT cells was performed as described previously [28 (link), 66 (link)].

Transfection with siRNA oligonucleotides was performed as described previously^{39 (link)}. Gene-specific siRNA oligonucleotides and AllStars negative control siRNA were obtained from Qiagen. The siRNA (80 pmol) was transfected into 1 × 10⁶ cells by electroporation using an EPI-2500 impulse generator (Fischer, Heidelberg, Germany) at 350 V for 10 ms. Treated cells were harvested after 24 h or 48 h.