IMAGING ONCOLYTIC ADENOVIRUSES SPREAD IN A 3D SYSTEM

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 研究病毒在3D培养中的传播。对病毒传播建模最相关的系统将是固体细胞团。如初步数据所示，我们已经能够通过将感染细胞和未感染细胞重组成Matrigel上的球体来成像固体团块中的单个感染细胞。我们将首先对感染Adegfuci的293细胞进行分析。我们首先用Adegfuci感染293细胞的单层，感染6小时后，感染的细胞将被胰酶消化。被感染的单细胞悬液将与过量的未感染的293细胞混合，并被接种到Matrigel基质上，以允许形成细胞聚集体和球体。这些球体将每天被监测绿色荧光细胞的出现，并将为任何最初显示一个受感染细胞的球体记录和编辑荧光Z堆栈蒙太奇。校园光学生物共享资源中有一台适合这些实验的共焦显微镜。这似乎可能是病毒在3D结构中的传播速度比在单层结构中更快，或者继发感染的细胞数量可能更多。也有可能确定最初的MOI对通过3D文化传播的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Study of viral spread in 3D cultures. The most relevant system for modeling viral spread would be in solid cell masses. As shown in the preliminary data, we have been able to image individual infected cells in solid masses by reconstituting infected and uninfected cells into spheres on Matrigel. We will first carry out analysis on AdEGFPuci-infected 293 cells. We will first infect a monolayer of 293 cells with AdEGFPuci, and 6 hr after infection, the infected cells will be trypsinized. The infected single cell suspensions will be mixed with an excess of uninfected 293 cells, and plated onto Matrigel matrices to allow formation of cell aggregates and spheres. The spheres will be monitored daily for appearance of green fluorescent cells and fluorescent Z-stack montages will be recorded and compiled for any sphere that initially show one infected cell. A confocal microscope suitable for these experiments is available in the campus optical biology shared resource. It seems possible that the rate of virus spread may be more rapid in 3D structures than in monolayer, or that the number of secondarily infected cells may be larger. It will also be possible to determine the effects of initial MOI on spread through 3D cultures.