Hideaki Matsuoka

Targeted delivery of a decoy oligodeoxynucleotide to a single ES cell by femtoinjection

UNLABELLEDnFemtoinjection has been proposed as a feasible approach for the targeted delivery of a decoy oligodeoxynucleotide (ODN) into a single ES cell for the study of transcription factor activity. Here, we evaluated the utility of decoy ODN delivery via femtoinjection in an ES cell model in which Venus fluorescent protein was expressed under the control of the tet-off system. Femtoinjection of a control decoy (Con-decoy) and a tetracycline response element decoy (TRE-decoy) into the cytoplasm had no apparent effect on Venus fluorescent protein expression; however, femtoinjection of the TRE-decoy into the nucleus successfully suppressed expression of the Venus fluorescent protein. We therefore conclude that it is feasible to suppress the activity of a transcription factor in a single ES cell by the delivery of a decoy ODN into the nucleus using the femtoinjection technique.nnnFROM THE CLINICAL EDITORnThe authors of this novel basic science study successfully demonstrate a femtoinjection technique to deliver a decoy oligodeoxynucleotide into a single ES cell.

A femto-injection technique for dynamic analysis of protein function in living embryonic stem cells

The potential of a femto-injection technique for use in analyzing protein dynamics in embryonic stem (ES) cells was investigated. First, we showed that fluorescent proteins could be injected in a quantitative fashion into individual mouse ES cells. Second, we demonstrated that the technique could identify functional differences between proteins by analyzing the effect of a nuclear localization signal on the behavior of glutathione S-transferase conjugated to green fluorescent protein. The analysis showed a clear difference in the distribution of the protein when the nuclear localization signal was present. Our results confirm that the non-destructive, quantitative and time controllable aspects of the technique provide considerable advantages for the analysis of protein behavior in living ES cells. To the best of our knowledge, this is the first report of the successful introduction of proteins into living ES cells by an injection technique.

Functional Control of Target Single Cells in ES Cell Clusters and Their Differentiated Cells by Femtoinjection

Microinjection is well recognized as an ideal but technically difficult method for the direct introduction of any molecules into single cells (King, 2004; Matsuoka et al., 2006). The difficulty depends on the cell size, intracellular structure, and the physical properties of the cell surface. Egg cells are an easy target because of their large size of 100–200 μm. Blastocysts are also a large target for the injection of embryonic stem (ES) cells (Fig. 1 (A)). For blastocysts, the tip diameter of an injector is 5 μm or greater. On the other hand, fibroblasts are as small as 20–30 μm but comparatively easy targets because their cell surface appears to be firm. Moreover they appear to be resilient against deep insertion of an injection capillary. By contrast, plant cells are larger than fibroblasts but much more difficult targets. Plant cells usually have large vacuoles in the intracellular space and therefore microinjection should be performed into a thin space between the cell membrane and the vacuole. Typical examples are tobacco cultured cells, BY-2, with a size of 40×80 μm (Fig. 1 (B)) and rice protoplasts with a diameter of 30–50 μm. By comparison, ES cells are extraordinarily difficult targets because of their small size (15–20 μm) (Fig. 1 (C)) and sticky cell surface. In fact, microinjection of a plasmid vector into ES cells was not successfully performed by a microinjection expert until our experiment performed in 2005 (Matsuoka et al., 2005). Before then, microinjection speed was no higher than 10 cells per h and the success rate was 7–8%. In the case of ES cells, the success rate was only 0.2%. Therefore, it was essential to increase the throughput of the method for the purpose of single-cell studies. Thus, we developed a useful robot that could support the microinjection operator; a single-cell manipulation supporting robot (SMSR) (Matsuoka et al., 2005). Using the SMSR, the injection speed increased to 100 cells per h and its success rate reached as high as 10%, even when operated by non-specialist personnel. The quantity of DNA ejected from the injection capillary was estimated to be no greater than 50 fg, and consequently the quantity of DNA actually introduced into a cell was in the fg range. Moreover, when an enhanced green fluorescent protein (EGFP) expression vector was injected into ES cells, the EGFP expression intensity changed in line with the varied concentration of the vector in the injection capillary. This method has been termed femtoinjection because it has enabled semiquantitative injection at the femtogram level (Matsuoka et al., 2007).

Single-Cell Injectoassay for ES Cell Engineering

It is important to analyze the intensity dependent action manners of differentiation associated genes in ES cell. For this purpose, single-cell injectoassay was performed by femto-injection method using a single-cell manipulation supporting robot. An Oct3/4 expression vector was introduced into mouse ES cell quantitatively and its effect was evaluated by a cell shape change indicator defined by the perimeter to radius ratio (PR ratio). In the cases of no injection control and the injection control, PR ratio was 7.3-8.0 until 72h. In contrast, when the vector concentration in the injector capillary was 300 ng/micro-L, the rate of differentiation was 33% (2/6). This rate further increased up to 86% (6/7) at 1200 ng/micro-L. These indicate the feasibility of the single-cell injectoassay.

Rapid and retrievable recording of big data of time-lapse 3D shadow images of microbial colonies

We formerly developed an automatic colony count system based on the time-lapse shadow image analysis (TSIA). Here this system has been upgraded and applied to practical rapid decision. A microbial sample was spread on/in an agar plate with 90u2009mm in diameter as homogeneously as possible. We could obtain the results with several strains that most of colonies appeared within a limited time span. Consequently the number of colonies reached a steady level (Nstdy) and then unchanged until the end of long culture time to give the confirmed value (Nconf). The equivalence of Nstdy and Nconf as well as the difference of times for Nstdy and Nconf determinations were statistically significant at pu2009<u20090.001. Nstdy meets the requirement of practical routines treating a large number of plates. The difference of Nstdy and Nconf, if any, may be elucidated by means of retrievable big data. Therefore Nconf is valid for official documentation.