Abstract
Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-016-9673-5) contains supplementary material, which is available to authorized users.
Highlights
For the last decades mouse embryonic stem cells have been considered to be one of the most interesting model systems for basic cellular and molecular studies and developmental research
We have found that when mouse embryonic stem (mES) cells are cultured in the serum-free 2i medium (containing two inhibitors, against the mitogenactivated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) and the glycogen synthase kinase 3 (GSK3), respectively [1]), chemical transfection is even more challenging as compared to when cells are cultured in standard culture media [2]
All transfection reagents significantly reduced Oct3/4 mRNA expression at both 24 and 48 h post-transfection, as compared to cells transfected with 100 nM scrambled small interfering RNA (siRNA) (Fig. 1a)
Summary
For the last decades mouse embryonic stem (mES) cells have been considered to be one of the most interesting model systems for basic cellular and molecular studies and developmental research. MES cells grow in tight clusters and are quite inefficiently transfected using chemical reagents. We have found that when mES cells are cultured in the serum-free 2i medium (containing two inhibitors, against the mitogenactivated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) and the glycogen synthase kinase 3 (GSK3), respectively [1]), chemical transfection is even more challenging as compared to when cells are cultured in standard culture media [2]. Replacement of serum by targeted inhibition of relevant signaling pathways reduces cellular heterogeneity as well as transcriptome and epigenome differences in mES cell cultures [3, 4]. In 2i medium PD0325901 inhibits the autocrine activation of the ERK1/2 pathway by fibroblast growth factor-4 (FGF4), shown to be instrumental for ES cell differentiation [5]. The cell cultures exhibit a more dense and uniform morphology, with negligible
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