Abstract
Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine.
Highlights
MethodsMouse AB1 ES cells were grown on mouse embryonic fibroblast (MEFs) feeders in standard ES culture medium
We examined transcriptional levels following electroporation of ES cells with several transcript fragments corresponding to different regions of Cdk[9]
Numerous studies have reported that RNAi gene silencing acts by targeting sense transcripts, our study shows that RNAi gene activation operates by targeting antisense transcripts
Summary
Mouse AB1 ES cells were grown on mouse embryonic fibroblast (MEFs) feeders in standard ES culture medium. ESCs were cultured on a feeder layer of Mitomycin C treated MEFs on 0.2% gelatin-coated cell culture dishes. Culture medium consisted of Dulbecco’s modified Eagle’s medium, 15% ES-grade fetal calf serum (FCS, Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Sigma), 100 U/ml penicillin and 0.1 mg/ml streptomycin and 1000 U/ml leukemia inhibitory factor (LIF). Mouse embryonic fibroblasts (MEFs) and human keratinocyte cell lines were cultured in DMEM containing 10% FCS. Ago1−/−, 3−/−, 4−/− ES cells were kindly provided by X. Electroporation was performed 1–2 days after plating (5 × 106 cells per plate) with 5 μg RNA using the Bio-Rad Gene Pulser apparatus (400 V, 250 F)
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