Collaborative Research: Development of Phytoplankton PCNA Antibodies for Use in Growth Studies: Emiliania huxleyi and Karlodinium micrum as Model Systems

增殖细胞核抗原（PCNA）是一种细胞周期标志物，可用于评估浮游植物的生长状况和估算原位生长速率。然而，由于缺乏针对浮游植物PCNA的有效抗体，其应用一直受到阻碍。近年来，浮游植物主要种类的pcna编码基因已被克隆和测序，这使得生产和鉴定有效的抗体成为可能。本项目将生产一套10种浮游植物PCNA抗体。与抗体生产公司合作，将使用合成肽生产三种通用藻类PCNA抗血清和三种甲藻特异性PCNA抗血清。这种安排将降低生产成本，并将为其他研究人员提供抗体供应。四种抗血清将基于细菌中各自的PCNA基因过表达纯化的PCNA，分别针对球石藻属赫斯利线虫(2)和鱼毒性甲酰鞭毛虫(2)生产。该项目将包括基于epifluorescence microscopy和流式细胞术，以E. huxleyi和K. micrum为模型系统，开发一种利用这些抗体的免疫荧光优化方案。PCNA在赫胥黎氏杆菌和微卡氏杆菌中的表达将采用Western Blots进行表征。PCNA与细胞分裂周期的关联将通过PCNA的免疫荧光标记，结合流式细胞术，基于dna的细胞周期分析来表征。将这些物种基于pcna的生长速率与培养中的实际生长速率（使用细胞计数）进行比较，将有助于评估新方法的准确性和精密度。共有10种多克隆抗体可供全球感兴趣的研究人员使用。这些PCNA抗血清将被证明对评估浮游植物种群的生长状态非常有用，并在研究人员自行决定使用各种检测格式来估计原位总生长率（GGR）。例子包括Western Blotting，免疫荧光，或潜在的酶联免疫吸附测定（ELISA）。PCNA抗体产品的可用性将促进未来的应用，包括研究两种模式物种和其他浮游植物的细胞周期和生长特性，以及PCNA方法在原位生长速率估计中的现场应用。该项目包括对研究生和本科生进行分子技术和流式细胞术分析方面的培训。两个参与机构之间的交流将使pi及其学生能够扩展他们的专业知识。研究结果将被整合到pi教授的课程中。该项目还将为学院继续致力于鼓励少数民族和女学生参与科学研究提供一个极好的机会。

Proliferating cell nuclear antigen (PCNA) is a cell cycle marker that is potentially useful for assessing phytoplankton growth status and estimating in situ growth rate. However, its application has been hindered by the lack of effective antibodies for phytoplankton PCNA. PCNA-encoding genes for major classes of phytoplankton have been cloned and sequenced recently, permitting production and characterization of effective antibodies. This project will produce a suite of ten phytoplankton PCNA antibodies. Three universal algal PCNA antisera and three dinoflagellate-specific PCNA antisera will be produced using synthetic peptides, in collaboration with an antibody producing company. This arrangement will reduce production costs and will provide a supply of antibody that can be made available to other researchers. Four antisera will be produced for the coccolithophorid species Eminiliania huxleyi (2) and the ichthyotoxic dinoflagellate Karlodinium micrum (2) based on purified PCNA overexpressed in bacteria from respective PCNA genes. This project will include the development of an optimized protocol for immunofluorescence using these antibodies, based on both epifluorescence microscopy and flow cytometry, with E. huxleyi and K. micrum as model systems. Expression of PCNA in E. huxleyi and K. micrum will be characterized using Western Blots. Association of PCNA with the cell division cycle will be characterized by means of immunofluorescence labeling of PCNA, coupled with flow cytometric, DNA-based analysis of the cell cycle. Comparisons of PCNA-based growth rates of these species to actual growth rates in culture (using cell counts) will allow assessment of the accuracy and precision of the new method. A total of 10 polyclonal antibodies will be available to interested researchers worldwide. These PCNA antisera will prove extremely useful for assessing the growth status of phytoplankton populations and for estimating in situ gross growth rates (GGR) using various detection formats atr the discretion of the researcher. Examples include Western Blotting, immunofluorescence, or potentially, enzyme linked immunoabsorbent assays (ELISA). The availability of PCNA antibody products will facilitate future applications including studies of the cell cycle and growth characteristics of the two model species and other phytoplankton, as well as field applications of the PCNA approach to in situ growth rate estimation. This project includes training for graduate and undergraduate students in molecular techniques and flow cytometric analyses. Travel between the two participating institutions will allow both PIs and their students to expand their expertise. Results of the research will be integrated in courses taught by the PIs. This project will also provide an excellent opportunity for the PIs to continue with their commitment to encourage minority and female students to participate in scientific research.