Semi-quantitative expression and knockdown of a target gene in single-cell mouse embryonic stem cells by high performance microinjection
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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.
KeywordsEmbryonic stem cell Gene expression control Knockdown RNAi Semi-quantitative microinjection
We thank H. Niwa of 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 funded 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.
- Matsuoka H, Saito M (2006) High throughput microinjection technology toward single-cell bioelectrochemistry. Electrochemistry 74:12–18Google Scholar
- Nishigori H, Tomura H, Tonooka N, Kanamori M, Yamada S, Sho K, Inoue I, Kikuchi N, Onigata K, Kojima I, Kohama T, Yamagata K, Yang Q, Matsuzawa Y, Miki T, Seino S, Kim MY, Choi HS, Lee YK, Moore DD, Takeda J (2001) Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects. Proc Natl Acad Sci USA 98:2575–2580CrossRefGoogle Scholar