Long-term cultivation of in vitro Apis mellifera cells by gene transfer of human c-myc proto-oncogene

Okumura, Naoko; Yoshida, Hitomi; Nishimura, Y.; Takahashi, Junichi; Matsuda, Satoru

doi:10.1007/s11626-011-9431-6

Cited by 19 publications

(18 citation statements)

References 11 publications

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“…Difficulty in adapting honey bees cells to in vitro conditions may be the result of selecting donor tissues whose age or origin is unsupportive of long-term growth. Recently, gene transfer technology has been used to evade these limitations, where insertion of the green fluorescent protein gene by lentivirus transduction [13] and the human c-myc proto-oncogene by lipofection [21] into embryonic honey bee cells was performed to demonstrate if activation of the transgenes was feasible and could promote long-term proliferation and survival. The latter method resulted in the establishment of a cell line that remained viable during an 8-month follow-up period; however, subsequent evidence to support claims of a continuous line has not been forthcoming.…”

Section: Introductionmentioning

confidence: 99%

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

2013

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A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

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Section: Introductionmentioning

confidence: 99%

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

2013

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“…RNA interference (RNAi) has been used for honey bee embryos in vivo to characterize the functioning of specific genes (16) and for genetic effects on morphological differentiation (17,18). Moreover, the cultivation of short-term (19 -21), long-term (22), and immortalized cell lines (23), and the expression of non-Apis genes in cultured embryonic cells (24) have opened up a new era for genetic manipulation of honey bee embryos.…”

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confidence: 99%

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica)

Mao

Han

et al. 2014

Molecular & Cellular Proteomics

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Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24 -48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, ␤-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48 -72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects. Molecular & Cellular Proteomics 13:

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“…Cell cultures were initiated from a specific stage of the honey bee embryo, the pre-gastrula stage, and cells remained mitotically active for more than three months [44], suggesting that honey bee embryos at this specific stage provide good starting material for long-term cultivation. Kitagishi Y et al engineered A. mellifera cells derived from honey bee embryos using the human c-myc proto-oncogene for their long-term cultivation [57]. The cell line, designated as MYN9, was successfully cultured for more than 100 generations over a period of more than eight months, suggesting that the human c-myc proto-oncogene was efficient for immortalization of honey bee cells.…”

Section: Continuous Cell Lines Derived From Hymenopteramentioning

confidence: 99%

“…While the AmE-711 cell line could have been contaminated during-or subsequent to-establishment, the prevalence of DWV in honey bees and vertical transmission of this virus [76] suggest that DWV was present in the embryos that were used as a starting material. Similarly, previously established primary cell lines as well as the genetically engineered continuous cell line MYN9 were also infected with DWV [47,57]. As vertical transmission of DWV results from virus adherence to the surface of the egg (i.e., transovum transmission) [76], it should be possible to remove the virus from the egg surface using a variety of published procedures [77].…”

Section: Cell Lines For Honey Bee Virus Studiesmentioning

confidence: 99%

Cell Lines for Honey Bee Virus Research

Guo

Goodman

Stanley

et al. 2020

Viruses

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With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of the virus load, there is an urgent need to protect honey bees (Apis mellifera) from fatal levels of virus infection and from the non-target effects of insecticides used in agricultural settings. A continuously replicating cell line derived from the honey bee would provide a valuable tool for the study of molecular mechanisms of virus-host interaction, for the screening of antiviral agents for potential use within the hive, and for the assessment of the risk of current and candidate insecticides to the honey bee. However, the establishment of a continuously replicating honey bee cell line has proved challenging. Here, we provide an overview of attempts to establish primary and continuously replicating hymenopteran cell lines, methods (including recent results) of establishing honey bee cell lines, challenges associated with the presence of latent viruses (especially Deformed wing virus) in established cell lines and methods to establish virus-free cell lines. We also describe the potential use of honey bee cell lines in conjunction with infectious clones of honey bee viruses for examination of fundamental virology.

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Long-term cultivation of in vitro Apis mellifera cells by gene transfer of human c-myc proto-oncogene

Cited by 19 publications

References 11 publications

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica)

Cell Lines for Honey Bee Virus Research

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