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

通过创造一种新的蜜蜂细胞系，这将有助于确定蜜蜂是如何死于微生物感染的，这将促进国家的繁荣，并增加国家的生物经济。世界范围内蜜蜂和其他传粉媒介的种群数量正在下降，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（Apis mellifera）群体的蜂群衰竭失调症，只是传粉媒介迅速丧失的一个戏剧性例子。虽然有大量的病毒感染蜜蜂，但尚不清楚这些病毒是如何影响蜜蜂数量下降的。蜜蜂细胞系对科学有益，因为它将使科学家能够确定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蜜蜂的第一个永生化细胞系。由此产生的永生化细胞系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，这项研究可能有助于了解其他传粉媒介物种的疾病动态，其中许多对美国农业很重要。这种细胞系可能是商业上可用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，包括代表性不足的学生。因此，这笔资金正在培养下一代科学领袖。研究人员将通过养蜂工作坊，以及通过农贸市场和视频纪录片开展的蜜蜂健康和研究意识活动，向公众分享他们的发现。由于缺乏长生不老的蜜蜂细胞系，蜜蜂病毒的研究受到了极大的阻碍。到目前为止，只有欧洲蜜蜂（Apis mellifera）建立了原代细胞培养。这些原代细胞培养很难建立和维持，并且有几个主要的缺点，包括存在多种驻留病毒，细胞分裂速度极慢，以及在达到衰老之前传代次数的限制。本研究将检验候选蜜蜂基因使蜜蜂胚胎细胞永生化的能力。这将通过在从胚胎分离的原代胚胎细胞中稳定表达候选基因并监测由此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，以便尝试获得代表各种细胞类型的永生化细胞系。选择具有最佳生长特性且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，任何存在的病毒将通过RNA干扰和单细胞克隆的结合来清除。由此产生的无病毒、永生的蜜蜂细胞系将提供给研究界。如果成功，这种方法也将为研究人员从其他蜜蜂物种中分离出永生细胞系提供模板。这项研究得到了美国国家科学基金会“共生、防御和自我认知”项目的支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.