Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells.
To derive an efficient system for gene silencing in human hematopoietic stem cells (HSCs) we modified a lentiviral vector for small interfering RNA (siRNA) delivery. For this purpose, an H1 promoter-driven siRNA expression cassette was introduced into a lentiviral vector, and the p53 mRNA was chosen as a target for siRNA-mediated gene silencing. Using the recombinant lentivirus we infected human cord blood-derived CD34+ cells and obtained a transfection efficiency of up to 50%, as determined by expression of enhanced green fluorescent protein (EGFP). In EGFP-positive long-term culture-initiating cell (LTC-IC)- and colony-forming unit cell (CFU-C)-derived cells, we observed a reduction of p53 mRNA of up to 95%. Importantly, this reduction remained stable during several weeks of cell culture. Furthermore, p53 gene silencing resulted in decreased p21 mRNA levels and reduced the sensitivity of CD34+ cells toward the cytotoxic drug etoposide. Thus, lentiviral delivery of siRNA can allow for efficient and stable gene silencing in human HSCs and will be very valuable for further gene function studies.
Department of Research, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland.
Tibor
T
Schomber
Christian P
CP
Kalberer
Aleksandra
A
Wodnar-Filipowicz
Radek C
RC
Skoda
D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D001483:Base Sequence; D017931:DNA Primers; D020868:Gene Silencing; D017930:Genes, Reporter; D016158:Genes, p53; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics / Q000493:pharmacokinetics*
D015703:Antigens, CD; D018952:Antigens, CD34; D017931:DNA Primers; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins