COLUMN FRACTION SCREENING OF CPMV PARTICLES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 需要能够在UV处理后分离和表征RNA填充的、空的和聚集的级分，以纯化含RNA的、非感染性的单个CPMV颗粒用于进一步的体内研究。 为了显示许多柱级分是否具有这些物理特性，目标是能够以有效的方式可视化从柱或蔗糖梯度收集的级分。 由于典型的柱运行产生20-100个级分，因此用于EM的各个CPMV级分的制备将显著受益于自动化。 然后需要分析TEM图像，以观察每个级分中的颗粒，以鉴定和定量RNA填充的衣壳、空衣壳、聚集的碎片和破碎的颗粒的比例。该程序将使单个灭活批次的CPMV纳米颗粒的体内使用可行性验证。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There is a need to be able to separate and characterize the RNA-filled, empty, and aggregated fractions following UV treatment in order to purify the RNA-containing, non-infectious single CPMV particles for further in vivo studies. In order to show whether the many column fractions have these physical characteristics, the goal is to be able to visualize fractions collected from columns or sucrose gradients in an efficient manner. Since a typical column run yields between 20-100 fractions, the preparation of individual CPMV fractions for EM would benefit significantly from automation. Analysis of the TEM images would then be needed, to view the particles in each fraction to identify and quantify the proportion of RNA-filled capsids, empty capsids, aggregated debris, and broken particles. This procedure would make the validation of individual inactivated lots of CPMV nanoparticles feasible for in vivo use.