Abstract
Single-cell manipulation supporting robot (SMSR) has enabled femtoinjection, a high-throughput and semi-quantitative microinjection in the range of femtogram (fg) DNA and other molecules. An enhanced green fluorescent protein (EGFP) gene expression vector can be introduced directly into mouse embryonic stem cells at 100 cells/h with a 10% success rate. The intensity of EGFP fluorescence in single ES cells or single colonies of ES cells increases as the concentration of an EGFP gene expression vector in the injection capillary increases from 1 to 50 ng/μL. On the other hand, the knockdown of EGFP gene expression can be demonstrated by femtoinjection of siRNA against EGFP or an shRNA expression vector using an EGFP expressing ES cell line. Femotoinjection can provide a useful method for quantitative analysis of transient gene expression in single cells using RNAi.
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Acknowledgments
We thank H. Niwa of the Center for Developmental Biology, RIKEN, Kobe, Japan for providing us with feeder free mouse ES cells (EB3 and G4-2) and plasmids (pCAG-IRES-DsRed and pCAG-cHA-IP-EGFP). This work was supported by funding to H. Matsuoka from CREST of Japan Science and Technology Agency on the research subject “The High Throughput Creation of Disease Model Cells and the Analysis of Their Function.” This work was partially supported by Strategic Research Promotion Program, the Ministry of Education, Culture, Sports, Science, and Technology, on the research subject “Development of Next Generation Bioresources.”
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Saito, M., Matsuoka, H. (2010). Semi-quantitative Analysis of Transient Single-Cell Gene Expression in Embryonic Stem Cells by Femtoinjection. In: Zhang, B., Stellwag, E. (eds) RNAi and microRNA-Mediated Gene Regulation in Stem Cells. Methods in Molecular Biology, vol 650. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-769-3_13
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DOI: https://doi.org/10.1007/978-1-60761-769-3_13
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