Skip to Content
MilliporeSigma
  • RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells.

RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells.

Scientific reports (2015-11-26)
Yvonne Diener, Marion Jurk, Britta Kandil, Yeong-Hoon Choi, Stefan Wild, Ute Bissels, Andreas Bosio
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cycloheximide, ≥90% (HPLC)
Sigma-Aldrich
MISSION® esiRNA, targeting mouse Prom1
Sigma-Aldrich
Cycloheximide, Biotechnology Performance Certified
Sigma-Aldrich
MISSION® esiRNA, targeting human TMEM131
Sigma-Aldrich
MISSION® esiRNA, targeting human PROM1
Sigma-Aldrich
Cycloheximide, from microbial, ≥94% (TLC)
Sigma-Aldrich
Fluorescein, for fluorescence, free acid
Sigma-Aldrich
8-Octanoyloxypyrene-1,3,6-trisulfonic acid trisodium salt, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
MISSION® esiRNA, targeting mouse Tmem131
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.0%, suitable for absorption spectrum analysis
Sigma-Aldrich
Dimethyl sulfoxide, JIS special grade, ≥99.0%
Sigma-Aldrich
Dimethyl sulfoxide, SAJ first grade, ≥99.0%
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5%
Sigma-Aldrich
Dimethyl sulfoxide, suitable for HPLC
Sigma-Aldrich
Phenylacetic acid, 99%
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology
Sigma-Aldrich
Dimethyl sulfoxide, puriss. p.a., ACS reagent, ≥99.9% (GC)
Sigma-Aldrich
Dimethyl sulfoxide, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Dimethyl sulfoxide, meets EP testing specifications, meets USP testing specifications
Sigma-Aldrich
Dimethyl sulfoxide, puriss. p.a., dried, ≤0.02% water
Sigma-Aldrich
Dimethyl sulfoxide, ACS reagent, ≥99.9%
Sigma-Aldrich
Dimethyl sulfoxide solution, 50 wt. % in H2O
Sigma-Aldrich
Cycloheximide solution, Ready-Made Solution, microbial, 100 mg/mL in DMSO, Suitable for cell culture
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Dimethyl sulfoxide, anhydrous, ≥99.9%
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma
Sigma-Aldrich
Dimethyl sulfoxide, PCR Reagent
Sigma-Aldrich
Dimethyl sulfoxide, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Dimethyl sulfoxide, Vetec, reagent grade, 99%