Open AccessA simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis
Author(s) -
Kiichi Watanabe,
Mikio Yoshiyama,
Gaku Akiduki,
Kakeru Yokoi,
Hiroko Hoshida,
Takumi Kayukawa,
Kiyoshi Kimura,
Masatsugu Hatakeyama
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0257770
Subject(s) - biology , ex vivo , transfection , green fluorescent protein , cell culture , gene expression , microbiology and biotechnology , in vitro , gene , in vivo , honey bee , genetics , botany
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.