Biotechnology Letters

, Volume 29, Issue 3, pp 341–350 | Cite as

Semi-quantitative expression and knockdown of a target gene in single-cell mouse embryonic stem cells by high performance microinjection

  • Hideaki Matsuoka
  • Soichiro Shimoda
  • Masakazu Ozaki
  • Hajime Mizukami
  • Meiri Shibusawa
  • Yohei Yamada
  • Mikako Saito
Original Research Paper

Abstract

Interactions of multiple genes and associated factors are involved in the differentiation and de-differentiation of embryonic stem (ES) cells. Quantitative analysis of these genes and factors is essential for the elucidation of their mechanism. To meet this requirement, we have investigated various experimental conditions for high performance microinjection into mouse ES cells. A speedy and rhythmic operation was found to be important and was accomplished robotically by using a single-cell manipulation technique and XY-address registrable culture dishes. Among many experimental parameters, the tip size of an injection capillary, the pressure condition, and the DNA concentration in the injection capillary were of critical significance. Their optimum values were 0.5–0.8 μm, 0.7 kgf/cm2 for 30 ms, and 1–100 ng/μl, respectively. Under these conditions, semi-quantitative control of the EGFP gene expression in mouse ES cells and its knockdown was successfully demonstrated.

Keywords

Embryonic stem cell Gene expression control Knockdown RNAi Semi-quantitative microinjection 

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Hideaki Matsuoka
    • 1
    • 3
  • Soichiro Shimoda
    • 1
  • Masakazu Ozaki
    • 1
  • Hajime Mizukami
    • 1
  • Meiri Shibusawa
    • 1
  • Yohei Yamada
    • 2
    • 3
  • Mikako Saito
    • 1
    • 3
  1. 1.Department of Biotechnology and Life ScienceTokyo University of Agriculture and TechnologyKoganei, Tokyo Japan
  2. 2.Chuo Precision Industrial Co. LtdChiyoda-ku, Tokyo Japan
  3. 3.CREST (Core Research for Evolutional Science and Technology)Japan Science and Technology AgencyKawaguchi, SaitamaJapan

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