Collaborative Research: Transient Protein Expression in Plant Tissue Culture

主要研究人员（pi）的总体目标是发展植物细胞和组织培养技术，这将允许快速评估植物中的外源基因表达。这项工作包括为植物细胞和组织培养的生物反应器的监测、控制和操作制定严格的原则。这些生物反应器的操作原理将用于将使用农杆菌共同培养进行DNA转化的瞬时表达系统与TMV载体或能够扩增DNA以生产蛋白质的单链DNA双病毒载体（基于烟草和玉米种子胚乳）的性能相关联，同时测试三种治疗性蛋白质的表达和积累：一种单克隆抗体（NR-LU-10 MAb），一种细胞因子（人生长激素，hGH）和一种血液转运蛋白（人转铁蛋白，hTR）。具体目标是：(1)制定植物细胞和根培养生物反应器生长的操作和控制策略；(2)开发和优化瞬时表达DNA的递送技术/条件；(3)利用生物反应器控制策略和优化的瞬时治疗性蛋白表达系统，评估植物细胞和组织培养中的异源转基因表达，并与具有相同蛋白的转基因植物的性能进行相关性研究。这是一个合作的GOALI项目，涉及三个具有不同互补科学和技术专长的pi。宾夕法尼亚州立大学首席PI Wayne Curtis是生物反应器技术领域的专家，博伊斯汤普森研究所和康奈尔大学的第二PI Hugh Mason是转基因植物领域的专家，孟山都综合蛋白质技术公司的Doug Russell是转基因植物蛋白质生产领域的专家。

The Principal Investigators (PIs)' overall objective is to develop plant cell and tissue culture technology, which will permit rapid evaluation of heterologous gene expression in plants. This work includes the development of rigorous principles for the monitoring, control and operation of bioreactors for plant cell and tissue culture. These bioreactor operational principles will be utilized to correlate performance of transient expression systems using Agrobacterium co-cultivation for DNA transformation and the TMV vector or a single-stranded DNA geminivirus vector capable of DNA amplification for protein production (based on tobacco and corn seed endosperm) while testing expression and accumulation of three therapeutic proteins: a monoclonal antibody (NR-LU-10 MAb), a cytokine (human growth hormone, hGH), and a blood transport protein (human transferrin, hTR). The specific objectives are to: (1) Develop operational and control strategies for bioreactor growth of plant cell and root culture, (2) Develop and optimize techniques / conditions for DNA delivery for transient expression, and (3) Utilize bioreactor control strategies and optimized transient therapeutic protein expression systems to evaluate heterologous transgene expression in plant cell and tissue culture, followed by correlation with performance in transgenic plants with the same proteins. This is a collaborative GOALI project involving three PIs with different complementary scientific and technological expertise. Wayne Curtis the primary PI at the Pennsylvania State University is an expert in the area of bioreactor technology, Hugh Mason the second PI at the Boyce Thompson Institute and Cornell University in the area of transgenic plants, and Doug Russell at the Integrated Protein Technologies of Monsanto in the area of production of proteins from transgenic plants.