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Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer.

ABSTRACT: While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the Fc?RI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of Fc?RI on FLMC, although on BMMC the expression of c-Kit came after Fc?RI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy.

SUBMITTER: Fukuishi N

PROVIDER: S-EPMC3616098 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer.

Fukuishi Nobuyuki N Igawa Yuusuke Y Kunimi Tomoyo T Hamano Hirofumi H Toyota Masao M Takahashi Hironobu H Kenmoku Hiromichi H Yagi Yasuyuki Y Matsui Nobuaki N Akagi Masaaki M

PloS one 20130403 4

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the F ...[more]

PMID: 23573287

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Project description:BackgroundNext generation sequencing based tumor tissue genotyping involves complex workflow and a relatively longer turnaround time. Semiconductor based next generation platforms varied from low throughput Ion PGM to high throughput Ion Proton and Ion S5XL sequencer. In this study, we compared Ion PGM and Ion Proton, with a new Ion S5XL NGS system for workflow scalability, analytical sensitivity and specificity, turnaround time and sequencing performance in a clinical laboratory.MethodsEighteen solid tumor samples positive for various mutations as detected previously by Ion PGM and Ion Proton were selected for study. Libraries were prepared using DNA (range10-40ng) from micro-dissected formalin-fixed, paraffin-embedded (FFPE) specimens using the Ion Ampliseq Library Kit 2.0 for comprehensive cancer (CCP), oncomine comprehensive cancer (OCP) and cancer hotspot panel v2 (CHPv2) panel as per manufacturer's instructions. The CHPv2 were sequenced using Ion PGM whereas CCP and OCP were sequenced using Ion Proton respectively. All the three libraries were further sequenced individually (S540) or multiplexed (S530) using Ion S5XL. For S5XL, Ion chef was used to automate template preparation, enrichment of ion spheres and chip loading. Data analysis was performed using Torrent Suite 4.6 software on board S5XL and Ion Reporter. A limit of detection and reproducibility studies was performed using serially diluted DLD1 cell line.ResultsA total of 241 variant calls (235 single nucleotide variants and 6 indels) expected in the studied cohort were successfully detected by S5XL with 100% and 97% concordance with Ion PGM and Proton, respectively. Sequencing run time was reduced from 4.5 to 2.5 hours with output range of 3-5 GB (S530) and 8-9.3Gb (S540). Data analysis time for the Ion S5XL is faster 1 h (S520), 2.5 h (S530) and 5 h (S540) chip, respectively as compared to the Ion PGM (3.5-5 h) and Ion Proton (8h). A limit detection of 5% allelic frequency was established along with high inter-run reproducibility.ConclusionIon S5XL system simplified workflow in a clinical laboratory, was feasible for running smaller and larger panels on the same instrument, had a shorter turnaround time, and showed good concordance for variant calls with similar sensitivity and reproducibility as the Ion PGM and Proton.

Dataset Information

Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer.

Publications

Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer.

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