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 FcεRI 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 FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. 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.
Highlights
Mast cells are known to be intimately involved in allergic responses through an aggregation of surface-expressed FceRI followed by a release of inflammatory mediators including histamine, prostaglandins and cytokines [1,2]
MacNeil et al indicated using a MAPK kinase 3 (MKK3)-knockout mouse that MKK3 is closely associated with the production of IL-4 in mast cells through the marked decrease of early growth response-1 binding to the IL-4 promoter region [12]
We reveal that FLMC have almost the same properties as bone marrowderived mast cells (BMMC), and expand the possibilities for characterization of proteins in mast cells in cases where gene manipulation causes an embryonic lethal phenotype
Summary
Mast cells are known to be intimately involved in allergic responses through an aggregation of surface-expressed FceRI followed by a release of inflammatory mediators including histamine, prostaglandins and cytokines [1,2]. P38MAPKa knockout mice are known to be embryonic lethal and die in mid-gestation with defects in placental and embryonic vasculature [16]. In such cases of fetal death by gene manipulation, the functional analysis of proteins in mast cells is virtually impossible because both ‘‘isolated’’ and ‘‘generated’’ mast cells are derived from adult tissues; they are isolated from lung [17], skin [18,19], tonsil [19] and peritoneal fluid [20], and are generated from bone marrow [21], peripheral blood [22] and umbilical cord blood [23]. We reveal that FLMC have almost the same properties as BMMC, and expand the possibilities for characterization of proteins in mast cells in cases where gene manipulation causes an embryonic lethal phenotype
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