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

Kazuyo Watanabe,Takumi Kayukawa,Hiroko Hoshida,Masatsugu Hatakeyama,Gaku Akiduki,Kiyoshi Kimura,Mikio Yoshiyama,Kakeru Yokoi,Vanessa Corby-Harris

doi:10.1371/journal.pone.0257770

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.

Highlights

Controlled in vitro cell systems, of both established cell lines and primary cultures, are valuable tools for understanding basic biological events by simplified host environment
Ex vivo cultured hemocyte cells are capable of inducing gene mis-expression and serve as an in vitro system for a gene functional analysis of the honey bee (Figs 1 and 10)
Primary cultured cells have been established from various tissues and organs in the honey bee, and these primary cell cultures are proven to be useful for viral studies and neuronal processes [15, 39, 40]

Summary

Introduction

Controlled in vitro cell systems, of both established cell lines and primary cultures, are valuable tools for understanding basic biological events by simplified host environment. Efforts to produce insect cell lines have resulted in the establishment of more than 1000 continuous cell lines from various insect species [1]. These cell lines are utilized for a broad range of biological research areas and the industrial mass production of recombinant proteins [2,3,4]. Researchers use cell lines derived from closely related or commercially available species as an alternative or develop their own cell cultures [5].

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