Collaborative Research: Transient Protein Expression in Plant Tissue Culture

首席调查员(PI)的总体目标是开发植物细胞和组织培养技术，这将使快速评估植物中异源基因的表达成为可能。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。这些生物反应器的工作原理将被用来关联利用农杆菌共培养进行DNA转化的瞬时表达系统的性能，以及能够进行DNA扩增以产生蛋白质的TMV载体或单链DNA双生病毒载体(基于烟草和玉米种子胚乳)，同时测试三种治疗性蛋白质的表达和积累：单抗(NR-LU-10MAb)、细胞因子(人生长激素，hGH)和血液运输蛋白(人转铁蛋白，HTR)。其具体目标是：(1)开发植物细胞和根培养生物反应器生长的操作和控制策略；(2)开发和优化用于瞬时表达的DNA传递技术/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来评估异源转基因在植物细胞和组织培养中的表达，以及与含有相同蛋白质的转基因植物的表现的相关性。这是一个合作的目标项目，涉及三个具有不同互补科技专长的私人投资机构。宾夕法尼亚州立大学的首席PI韦恩·柯蒂斯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位PI休·梅森是转基因植物领域的第二位PI，孟山都综合蛋白质技术公司的道格·罗素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.