title: Nonviral transfection of leukemic primary cells and cells lines by siRNA-a direct comparison between Nucleofection and Accell delivery.
abstract: Transient downregulation of genes in vitro employing short interfering RNA (siRNA) is a time-honored approach to study gene function. A crucial prerequisite to obtain a downregulation is an efficient and nontoxic delivery of the siRNA into the target cells. However, this has proven difficult to accomplish, particular in cells in suspension. Thus, there is a need for a systematic evaluation of different methodologies to identify the most suitable protocol. We compared Nucleofection with Accell, a novel nonviral-based delivery system in the setting of leukemic blasts from patients with myeloid leukemias. Two cell surface proteins, human inhibitory C-type lectin-like receptor and CD96, both believed to be associated with leukemic stem cells, were chosen as target genes. Accell not only yielded higher transfection rates, but also retained superior cell viabilities for both cell lines and primary leukemic cells. Thus, transfection efficiencies in primary cells after Accell delivery was 85% (range, 71-97%) compared to 38% (23-65%) using Nucleofection for siRNA delivery. Preliminary studies of clonal growth of primary acute myeloid leukemia cells indicated growth inhibition after siRNA transfection. Our results reveal that Accell delivery is suitable for nonviral transfection of cells in suspension, including primary leukemic cells. These data should provide a platform for further studies of genes involved in early leukemogenesis.
authors.affiliation: Department of Hematology, Aarhus University Hospital, Denmark.
authors.forename: Hanne Østergård
authors.initials: HØ
authors.lastname: Larsen
authors.forename: Anne Stidsholt
authors.initials: AS
authors.lastname: Roug
authors.forename: Katrine
authors.initials: K
authors.lastname: Nielsen
authors.forename: Claus Svane
authors.initials: CS
authors.lastname: Søndergaard
authors.forename: Peter
authors.initials: P
authors.lastname: Hokland
mesh_terms: D015703:Antigens, CD / Q000235:genetics; D045744:Cell Line, Tumor; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics; D007938:Leukemia / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D008722:Methods; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000494:pharmacology / Q000627:therapeutic use; D014162:Transfection / Q000379:methods* / Q000592:standards; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; C116131:CD96 antigen; D037181:Lectins, C-Type; D034741:RNA, Small Interfering