title: RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells.
abstract: Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5'- and 3'-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs.
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Diener
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Jurk
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Kandil
authors.affiliation: Heart Center of the University of Cologne, Department of Cardiothoracic Surgery, Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Yeong-Hoon
authors.initials: YH
authors.lastname: Choi
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Wild
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Bissels
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bosio
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000071916:AC133 Antigen; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D018274:Electroporation; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D017493:Leukocyte Common Antigens / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D010455:Peptides / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000071916:AC133 Antigen; D015703:Antigens, CD; D005456:Fluorescent Dyes; D006023:Glycoproteins; D008081:Liposomes; D010455:Peptides; D011108:Polymers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017493:Leukocyte Common Antigens