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A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis.

ABSTRACT: Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace's insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

SUBMITTER: Watanabe K

PROVIDER: S-EPMC8460014 | biostudies-literature |

REPOSITORIES: biostudies-literature

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In vivo and in vitro infection dynamics of honey bee viruses.

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| S-EPMC4770293 | biostudies-literature

A simple and robust LC-ESI single quadrupole MS-based method to analyze neonicotinoids in honey bee extracts.

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| S-EPMC6838890 | biostudies-literature

Gelam honey potentiates ex vivo corneal keratocytes proliferation with desirable phenotype expression.

Project description:This study aimed to evaluate the effects of Gelam honey on corneal keratocytes proliferative capacity and phenotypic characterization via MTT assay, gene expression and immunocytochemistry.Corneal keratocytes from New Zealand white rabbits were cultured in basal medium (BM) and serum enriched medium (BMS). Serial dilutions of Gelam honey (GH) were added to both media and cells were cultured until passage 1. MTT assay was performed on corneal keratocytes in both media to ascertain the optimal dose of GH that produced maximum proliferation.Gelam honey at the concentration of 0.0015% in both media showed the highest proliferative capacity with no morphological changes compared to their respective controls. The gene expression of aldehyde dehydrogenase (ALDH), a marker for quiescent keratocytes and vimentin, a marker for fibroblast, were higher in the GH enriched groups. The alpha smooth muscle actin (?-SMA) expression, marker for myofibroblast, was lower in GH treated groups compared to the controls. Immunocytochemistry results were in accordance to the gene expression analyses.Gelam honey at a concentration of 0.0015% promotes ex vivo corneal keratocytes proliferation while retaining desirable phenotype expression. The results serve as a basis for the development of Gelam honey as a potential natural product in promoting corneal wound healing.

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| PRJNA1127320 | ENA

Functional diversity within the simple gut microbiota of the honey bee.

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Gelam honey promotes ex vivo corneal fibroblasts wound healing.

Project description:This study evaluated the effects of Gelam honey (GH) on ex vivo corneal fibroblast ulcer model via wound healing assay, gene expression and immunocytochemistry. Corneal fibroblasts from New Zealand white rabbits were culture expanded. The corneal fibroblast wound healing capacity was observed by creating a circular wound onto confluent monolayer cells cultured in basal medium (BM), BM with GH, serum-enriched basal medium (BMS) and BMS with GH respectively. Wound healing assay and phenotypic characterization of the corneal fibroblast were performed at different stages of wound closure. Expression of aldehyde dehydrogenase (ALDH), vimentin, α-smooth muscle actin (α-SMA), lumican, collagen I and matrix metalloproteinase 12 (MMP 12) were measured at day 1, day 3 and complete wound closure day. Corneal fibroblast cultured in BMS with GH demonstrated the fastest wound closure, at day 5 post wounding. The gene expressions of ALDH and vimentin were higher than control groups while α-SMA expression was lower, in GH enriched media. The expressions of lumican, collagen I and MMP 12 were also higher in cells cultured in GH enriched media compared to the control groups. GH was shown to promote in vitro corneal fibroblast wound healing and may be a potential natural adjunct in the treatment of corneal wound.

| S-EPMC6874633 | biostudies-literature

Changes in chemokine receptor expression of regulatory T cells after ex vivo culture.

Project description:By controlling and limiting inflammatory conditions, naturally occurring regulatory T cells (Tregs), defined as circulating CD4(+)CD25(bright)FoxP3(+) cells, play critical roles in maintaining tolerance and preventing autoimmunity and thus have tremendous potential for adoptive immunotherapy. Because they represent a scanty subset of the CD4(+) T-lymphocyte subset, several approaches have been developed to isolate and expand ex vivo polyclonal Tregs. However, one limitation of the functional analyses performed on these cultured Tregs is the incomplete characterization of their tissue-trafficking properties. As this aspect provides crucial information for their therapeutic effects, we have here explored the chemokine receptor expression profile and function of Tregs cultured ex vivo with validated expansion protocols. Our data show that ex vivo cultured Tregs retained the expression of CCR7 but dramatically downregulated CCR5 as compared with freshly isolated Tregs. The differential chemokine receptors expression pattern corroborated with their respective steady state messenger RNA expression and also with their migration toward specific chemokines. Our analyses suggest that ex vivo cultured Tregs may display impaired or suboptimal migration to the inflamed tissues releasing RANTES and MIP-1? chemokines.

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Honey bee: Apis mellifera syriaca

Project description:Apis mellifera syriaca is the native honeybee subspecies of Jordan and much of the Middle East. It expresses behavioral adaptations to a regional climate with very high temperatures, nectar dearth in summer, attacks of the Oriental wasp Vespa orientalis and in most cases it is resistant to varroa mites. The Thorax control sample of A. m. syriaca in this experiment was originally collected and stored since 2001 from Wadi Ben Hammad a remote valley in the southern region of Jordan. Using morphometric and Mitochondrial DNA markers it was proved that bees from this area had show higher similarity than other samples collected from the Middle East as represented by reference samples collected in 1952 by Brother Adam. The samples L1-L5 are collected from the National Center for Agricultural Research and Extension breading apiary which was originally established for the conservation of Apis mellifera syriaca. Goal was to use the genetic information in the breeding for varroa resistant bees and to determine the successfulness of this conservation program. Project funded by USAID-MERC grant number: TA-MOU-09-M29-075.

2016-02-01 | GSE43554 | GEO

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Project description:Honey bee drones, queens and workers have vastly different phenotypes. Here we profile the the expression level of mRNAs and microRNAs of honeybee, drones, queens and workers at the L5 larval stage (91 hours +/- 1).

2015-09-06 | GSE61253 | GEO

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2016-02-01 | GSE45464 | GEO

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