Kazumi Hakamada

Evaluation method for gene transfection by using the period of onset of gene expression and cell division

Using single-cells time-lapse analysis, we investigated the mechanism of gene expression using nine transfection reagents. Although onset of gene expression occurred after cell division by all reagents, 91.6% periods, which depended on onset and cell division, had statistical significance. Evaluation of those periods is useful for elucidating mechanism of transfection.

Onset timing of transient gene expression depends on cell division

By using the time lapse of both phase-contrast and fluorescent images, we examined the morphology of cells and the dynamics of gene expression (EGFP). We applied k-means clustering to the time course data of fluorescent intensity of EGFP and successfully found four subpopulations. Discriminating the appropriate clusters and investigating the details of them, we found that almost all cells express the transfected gene after cell division and also found there is a strong correlation between onset timing and cell division. This result suggested that it is possible to normalize the dynamics of gene by arranging the onset timing of gene expression or by arranging the cell division.

Motility behavior of rpoS-deficient Escherichia coli analyzed by individual cell tracking.

Motility is one of the most extensively studied cellular events conducted by bacteria, including Escherichia coli. A motility agar plate assay showed that deletion of the rpoS gene enhanced the apparent motility of the E. coli BW25113 strain, which inherently had negligible motility compared to wild-type E. coli strains, such as MG1655, with no effect on cell growth. This enhancement of motility was accompanied by drastic up-regulation of genes involved in the formation and rotation of flagella. Furthermore, an individual cell motility assay showed that the population of ΔrpoS cells had bimodal motility character, and that a minority of this population exhibited a much higher motility rate. These results support a view that a minority population contributes to increasing in apparent motility of the whole population of ΔrpoS cells.

Transfection Microarrays for High-Throughput Phenotypic Screening of Genes Involved in Cell Migration

Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening.

Identification of kinases and regulatory proteins required for cell migration using a transfected cell-microarray system

BackgroundCell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy. Previously, we developed a simple chip, based on transfected-cell microarray (TCM) technology, for the identification of genes related to cell migration. In the present study, we used the TCM chip for high-throughput screening (HTS) of a kinome siRNA library to identify genes involved in the motility of highly invasive NBT-L2b cells.ResultsWe performed HTS using TCM coupled with a programmed image tracer to capture time-lapse fluorescence images of siRNA-transfected cells and calculated speeds of cell movement. This first screening allowed us to identify 52 genes. After quantitative PCR (qPCR) and a second screening by a conventional transfection method, we confirmed that 32 of these genes were associated with the migration of NBT-L2b cells. We investigated the subcellular localization of proteins and levels of expression of these 32 genes, and then we used our results and databases of protein-protein interactions (PPIs) to construct a hypothetic but comprehensive signal network for cell migration.ConclusionsThe genes that we identified belonged to several functional categories, and our pathway analysis suggested that some of the encoded proteins functioned as the hubs of networks required for cell migration. Our signal pathways suggest that epidermal growth factor receptor (EGFR) is an upstream regulator in the network, while Src and GRB2 seem to represent nodes for control of respective the downstream proteins that are required to coordinate the many cellular events that are involved in migration. Our microarray appears to be a useful tool for the analysis of protein networks and signal pathways related to cancer metastasis.

A system for analyzing the event timing profile of single cells by using a model of neurite maturation in PC12D cells

Nerve growth factor (NGF)-induced neurite maturation in PC12D cells involves neuritogenesis and neurite outgrowth. Actions of compounds affecting the neurite maturation are sometimes invisible behind the individually variable events in nature even in the clonal population. In this study, we designed a time-lapse imaging system to determine the timing of each event in individual PC12D cells. To examine the system, we analyzed the effect of staurosporine on the neurite maturation in PC12D cells. By using the system, we obtained four event timing data sets (stimulation by NGF with and without staurosporine at the concentrations of 0.01, 0.1, and 1 microM). A permutation test of these data sets revealed that staurosporine caused an early induction of neurite outgrowth during neurite maturation in PC12D cells. These results suggest that the time-lapse imaging system to determine the timing of each event in individual cells can provide a novel insight into features of a cell mass by single-cell analysis and is expected to be utilized for profiling of compounds that can serve as candidate drugs.

Evaluating a time-delay of hydrogen production quantitatively in photosynthetic bacteria for stabilizing intermittency

Renewable energy is regarded as a clean energy source but has some problems, one of which is intermittency. To reduce this, the time-delay of hydrogen production by photosynthetic bacteria can be effective. In this study, we qualitatively evaluated the time-delay of hydrogen production by photosynthetic bacteria under various irradiation conditions, and we also quantitatively evaluated it by fitting the experimental data and the hydrogen production model with a genetic algorithm. As a result of model fitting, we found that the relationship between the lengths of the optimized time-delay of hydrogen production by photosynthetic bacteria and the amount of light irradiation is linear. And we also found that the time-delay of hydrogen production by photosynthetic bacteria had an upper limit under low light intensity. We have suggested the existence of an energy store mechanism in photosynthetic bacteria.

Evaluation of promoter activity by using single cell time course analysis

By using the time lapse of both phase-contrast and fluorescent images of single cells, we investigated the promoter activity. HeLa cells were transfected with EGFP, which encodes EGFP under the control of CMV promoter or cMyc binding site, in the presence of Lipofectamin™ LTX. After transfection with EGFP, phase-contrast and fluorescent images of cells in each well were recorded at intervals of 10min for 24 hours by using a Programmable Cellular Image Tracer. The strong correlation between cell division and the onset timing of gene expression was not influenced by the transfected gene. On the contrary in the case of pMycEGFP, the intercept of regression line was 54.685 min and that was 562.82 min in the case of pCMVEGFP. Intercept means the total requirement time from nuclear entry to folding of EGFP. It suggested that the activity of promoter of CMV was about 10 fold higher than that of cMyc promoter. This result suggested that the activity of promoters were comparable by using single cell time course analysis. This result also indicates that by using single cell time course analysis, we could evaluate the time of every step by arranging transfection reagent, promoter, and gene.