多活性猪猪Bac转座酶介导的秋粘虫，蜉蝣属果蝇蛆中的种系转化
September 23rd, 2021
秋粘虫 Spodoptera frugiperda的种系转化是使用过度活跃的 piggyBac 转座酶的mRNA实现的。
该协议显示了使用基于piggyBac的转化系统生产转基因秋粘虫。我们希望这些方法将有利于改善转化，不仅在秋粘虫中，而且在鳞翅目昆虫中。这里描述的方法使鳞翅目害虫昆虫的种系转化效率很高。
演示该程序的将是我实验室的博士后研究员Xien Chen博士。首先将12只雄性蛹和12只雌性蛹放在一个盒子里。将10%蔗糖溶液放入盒子中喂养成人。
用纸巾盖住盒子。在闭合后的第二天，将成虫暴露在强光下过夜。在关闭后的第三天，将成年人转移到黑暗的地方。
在过度放置后两小时内收集胚胎。在纸巾上加入一滴水，将新鲜鸡蛋放在冰上的培养皿中。用细刷子将鸡蛋轻轻地转移到载玻片上，并在载玻片上逐个对齐。
在显微注射之前，保持载玻片，将鸡蛋放在冰上。使用自动显微注射器的补偿压力，将每微升200纳克小猪Bac表达质粒，每微升200纳克超活性猪Bac转座酶mRNA和无菌蒸馏水的混合物注入鸡蛋中。将注射的卵保持在27加或零下1摄氏度，60±10%相对湿度，直到孵化。
用人工饲料喂养孵化的幼虫，并在早期凝乳和星形阶段将每个幼虫转移到一个小杯子中。收集蛹，并将大约150只雌性和150只雄性蛹放在笼子里。在笼子里挂几条30厘米×15厘米大小的纸巾，以收集鸡蛋。
每天收集带有鸡蛋的纸巾，并将鸡蛋保持在适当的条件下直到孵化。将新生儿幼虫保持在四摄氏度下五分钟以使其固定，然后将其转移到冰冷的盘子上。在荧光体视显微镜下对固定的新生儿幼虫进行筛查。
选择EGFP阳性的新生儿，在大约两周内以27摄氏度或零下一摄氏度将其提升到蛹阶段。使用1%琼脂糖凝胶检测体外合成的过度活跃的piggyBac转座酶mRNA。PDS注射的胚胎没有显示EGFP信号，而表达EGFP的质粒和过度活跃的piggyBac转座酶mRNA注射的胚胎显示出EGP荧光，表明piggyBac构建是成功的。
从G0成虫自交产生的卵中获得的G1幼虫显示出强烈的EGFP信号。使用从EGFP阳性幼虫中分离的基因组DNA作为模板，成功扩增了含有启动子和EGFP片段的片段。然而，当从野生型幼虫中分离出基因组DNA作为模板时，没有检测到相同的片段。
用于显微注射的卵子应尽可能新鲜，并且在显微注射之前必须始终保持在冰上以减缓其发育。该技术为研究人员回答害虫功能基因组学中的问题并为这种全球害虫开发遗传控制方法铺平了道路。
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概述
本研究展示了一种成功的禾谷螟（Spodoptera frugiperda）生殖系转化协议，利用基于piggyBac的转化系统。该方法承诺在鳞翅目害虫中提高转化效率，并增强这些生物的功能基因组学研究。
关键研究组成部分
研究领域
- 害虫的基因工程
- 转化生物学技术
- 功能基因组学应用
背景
- 禾谷螟是一种重要的农业害虫。
- piggyBac转座子系统以其基因递送的效率而闻名。
- 改进生殖系转化对于害虫控制策略至关重要。
使用的方法
- 用piggyBac质粒和mRNA进行卵的微注射
- Spodoptera frugiperda作为生物模型
- 通过荧光显微镜筛选转基因幼虫
主要结果
- 转基因幼虫中EGFP的成功表达显示了piggyBac系统的功能性。
- 通过基因组DNA扩增验证了EGFP表达，区分转基因和野生型幼虫。
- 该转化技术被证明在生产转基因害虫方面是有效的。
结论
- 研究建立了一种用于Spodoptera frugiperda生殖系转化的稳定方法。
- 研究结果有助于推进农业害虫的基因控制措施。
常见问题
Executive Industry Relevance
Establishing efficient germline transformation in agricultural pests like fall armyworm enables functional genomics for target validation and mechanistic de-risking in pest control pipelines. This protocol supports early discovery by providing a scalable system to interrogate gene function and assess phenotypic outcomes in a disease-relevant insect model. Successful transgenesis facilitates downstream screening and biomarker discovery for genetic pest management strategies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables functional interrogation of pest genes to validate targets for genetic control strategies.
- Operational Value: Provides a reproducible system to generate transgenic lines for consistent target assessment.
- Predictive Value: Supports mechanistic de-risking by linking gene disruption to phenotypic effects in a pest model.
Screening & Assay Development
- Scientific Value: Generates fluorescent reporter lines for high-throughput screening of genetic perturbations.
- Operational Value: Enables standardized PCR-based genotyping and TAIL-PCR for insertion site validation.
- Assay Readiness: Produces stable transgenic strains suitable for compound screening and phenotypic assays.
Translational & Preclinical Research
- Translational Continuity: Links discovery-phase gene validation to preclinical evaluation of genetic control mechanisms.
- Risk-Adjusted Advancement: Facilitates assessment of gene essentiality and phenotypic impact before field application.
- Biomarker Alignment: Supports identification of genetic markers associated with susceptibility or resistance traits.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by enabling target validation through transgenesis, supporting lead identification via phenotypic screening, and informing preclinical decisions through stable genetic models.
- Discovery Biology: Supports hypothesis testing by enabling gene knockout or overexpression in fall armyworm to assess target relevance.
- Screening: Generates standardized transgenic lines for reproducible screening of genetic or chemical modulators.
- Analytics: Provides quantitative PCR and fluorescence readouts to measure transgene integration and expression levels.
- Translational Research: Connects gene function discovery to preclinical evaluation of genetic pest control approaches.
- Enterprise Reuse: Establishes a reusable transgenesis platform applicable across lepidopteran pest species for portfolio-wide target validation.
Operational & Enterprise Impact
- Scientific Value: Enhances target validation confidence through direct genetic manipulation in a pest model.
- Operational Value: Delivers a scalable, reproducible transgenesis system with standardized microinjection and screening protocols.
- Strategic Value: Improves go/no-go decisions by reducing biological uncertainty in target essentiality.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on in vivo phenotypic validation in a pest model.
Implementation Considerations
- Requires expertise in insect embryology and microinjection techniques.
- Needs automated microinjector, fluorescence stereo microscope, and PCR equipment for validation.
- Demands standardized protocols for embryo collection, injection conditions, and transgenic screening across teams.
- Requires adaptation of temperature, humidity, and diet protocols for different lepidopteran species.
- Dependent on embryo quality and freshness, with microinjection efficiency influenced by developmental timing.
