Collaborative Research: EAGER: Establishing immortalized cell lines from the European honey bee, Apis mellifera

合作研究：EAGER：建立来自欧洲蜜蜂（Apis mellifera）的永生化细胞系

• 批准号: 2024026
• 负责人: Carol Fassbinder-Orth
• 金额: $ 12.27万
• 依托单位: Creighton University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2024026&HistoricalAwards=false
• 关键词: Collaborative; Research; EAGER; Establishing; immortalized

This EAGER will advance the national prosperity and add to national bioeconomy by creating a new bee cell line that will help identify how bees succumb to microbial infections. Populations of bees and other pollinators are in decline worldwide, which has major implications for ecosystem health and agricultural production interests. Colony collapse disorder, involving colonies of the European honey bee, Apis mellifera, is just one dramatic example of rapid loss of pollinators. Although a large number of viruses infect bees, it is not known how these viruses impact bee decline. A bee cell line would benefit science because it would allow scientists to identify how bee viruses infect and kill bees. The goal of this research is to establish and characterize the first immortalized cell lines from Apis mellifera. The resulting immortalized cell line(s) will provide an invaluable resources for the virology research field. In addition to helping the honey bee scientific community, this research may help understand disease dynamics in other pollinator species, many of which are important for US agriculture. This cell line may be the basis for commercially-available testing kits to identify or track bee diseases. The research uses funding to train graduate and undergraduate students, including under-represented students. As such this funding is training the next generation of leaders in science. Researchers will share their findings to the public through the use of beekeeping workshops, and honey bee health and research awareness activities through farmer’s markets and video documentaries. The lack of immortalized bee cell lines has greatly hampered research on bee viruses. Thus far, only primary cell cultures have been established from Apis mellifera, the European honey bee. These primary cell cultures are difficult to establish and maintain, and suffer from several major drawbacks including the presence of multiple resident viruses, extremely slow cell division rates, and limitations on the number of passages before reaching senescence. This research will examine the ability of candidate honey bee genes to immortalize A. mellifera embryonic cells. This will be accomplished by stably expressing candidate genes in primary embryonic cells isolated from embryos and monitoring growth and survival of the resulting cells. Primary cells isolated from different developmental stages of the embryo will be used, in order to attempt to obtain immortalized cell lines that represent various cell types. Cell clones with optimum growth characteristics that are able to be stably passaged will be selected. The resulting immortalized cell lines will be screened for resident viruses by deep sequencing, and any viruses present will be cleared by using a combination of RNA interference and single cell cloning. The resulting virus-free, immortalized honey bee cell lines will be made available to the research community. If successful, this approach will also provide a template for researchers to isolate immortalized cell lines from other bee species. This research was supported by the Symbiosis, Defense and Self-Recognition program of the National Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

这种渴望将通过创造一种新的蜜蜂细胞系来促进国家繁荣并增加国家生物经济，该细胞系将有助于识别蜜蜂如何屈服于微生物感染。蜜蜂和其他传粉者的数量在全球范围内正在下降，这对生态系统健康和农业生产利益具有重大影响。涉及欧洲蜜蜂蜂群的蜂群崩溃障碍，只是传粉者迅速丧失的一个戏剧性例子。虽然大量的病毒感染蜜蜂，但这些病毒是如何影响蜜蜂衰退的尚不清楚。蜜蜂细胞系将使科学受益，因为它将允许科学家识别蜜蜂病毒是如何感染和杀死蜜蜂的。本研究的目的是建立和鉴定第一个来自意大利蜜蜂的永生化细胞系。由此产生的永生化细胞系(S)将为病毒学研究领域提供宝贵的资源。除了帮助蜜蜂科学界，这项研究还可能有助于了解其他传粉物种的疾病动态，其中许多对美国农业很重要。该细胞系可能是商业化的检测试剂盒的基础，以识别或跟踪蜜蜂疾病。这项研究使用资金来培训研究生和本科生，包括代表不足的学生。因此，这笔资金正在培养科学领域的下一代领导者。研究人员将通过利用养蜂讲习班向公众分享他们的发现，并通过农贸市场和视频纪录片开展蜜蜂健康和研究意识活动。缺乏永生的蜜蜂细胞系极大地阻碍了对蜜蜂病毒的研究。到目前为止，只建立了欧洲蜜蜂意大利蜜蜂的原代细胞培养。这些原代细胞培养很难建立和维持，并存在几个主要缺陷，包括存在多个驻留病毒，细胞分裂速度极慢，以及在达到衰老之前传代的数量限制。这项研究将检验候选蜜蜂基因使意大利蜜蜂胚胎细胞永生的能力。这将通过在从胚胎中分离的初级胚胎细胞中稳定表达候选基因并监测产生的细胞的生长和存活来实现。将使用从胚胎不同发育阶段分离的原代细胞，以试图获得代表各种细胞类型的永生化细胞系。将选择生长特性最佳、能够稳定传代的细胞克隆。由此产生的永生化细胞系将通过深度测序筛选驻留病毒，并将使用RNA干扰和单细胞克隆相结合的方法清除任何存在的病毒。由此产生的无病毒、永生化的蜜蜂细胞株将提供给研究界。如果成功，这种方法还将为研究人员提供一个模板，将永生细胞系从其他蜜蜂物种中分离出来。这项研究得到了国家科学基金会共生、防御和自我认可计划的支持。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。