Universität zu Köln

Diener, Yvonne, Jurk, Marion, Kandil, Britta, Choi, Yeong-Hoon, Wild, Stefan, Bissels, Ute and Bosio, Andreas (2015). RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells. Sci Rep, 5. LONDON: NATURE PUBLISHING GROUP. ISSN 2045-2322

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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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Diener, Yvonne UNSPECIFIED UNSPECIFIED UNSPECIFIED Jurk, Marion UNSPECIFIED UNSPECIFIED UNSPECIFIED Kandil, Britta UNSPECIFIED UNSPECIFIED UNSPECIFIED Choi, Yeong-Hoon UNSPECIFIED UNSPECIFIED UNSPECIFIED Wild, Stefan UNSPECIFIED UNSPECIFIED UNSPECIFIED Bissels, Ute UNSPECIFIED UNSPECIFIED UNSPECIFIED Bosio, Andreas UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-386780
DOI:	10.1038/srep17184
Journal or Publication Title:	Sci Rep
Volume:	5
Date:	2015
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	2045-2322
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language EFFICIENT TRANSFECTION; POLYETHYLENIMINE; NANOPARTICLES; INTERFERENCE; ACTIVATION; EXPANSION; VIVO Multiple languages Multidisciplinary Sciences Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/38678