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.
Highlights
Destabilisation of the endosomal membrane by the cationic lipids
Haematopoietic stem and progenitor cells (HSPCs) were transfected with ten different reagents covering three classes of chemical transfection methods
Both have been described for transfection of HSPCs, but PEI 2 K showed a higher efficiency than ExGen 500, which is based on a 22 K, linear PEI molecule[13]
Summary
The so-called proton-sponge effect is thought to mediate the endosomal escape: During acidification of the endosome, the polymers bind large amounts of incoming protons. Physical transfection methods such as electroporation bypass the endosomal pathway by creating transient pores in the cell membrane and directly delivering nucleic acids into the cytosol[11]. Some reagents suggested a highly efficient transfection with a non-targeting, fluorescently labelled siRNA, we observed no target knockdown using functional siRNAs. In contrast, electroporation allowed an efficient delivery of RNAs into HSPCs. Both, protein knockdown using siRNAs as well as high and persistent protein overexpression with mRNA molecules could be achieved
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