MRI: Acquisition of a Fluorescence Stereomicroscope to Enhance Undergraduate Research at Colorado College

这项主要研究仪器拨款支持获得荧光立体显微镜和成像系统，以加强科罗拉多学院生物系Darrell J. Killian博士和Nancy N.Huang博士的研究项目。活体模型生物荧光分子的实时可视化已经成为一项不可或缺的技术，因为它可以直接观察和记录基因活性的变化，细胞内蛋白质的定位，以及在发育过程中对细胞和组织的跟踪。生物学中的一个重要问题是基因活动的变化如何决定性别特异性发育。Killian实验室对流行的模式生物秀丽隐杆线虫（C. elegans）中影响性别特异性发育的基因的鉴定和表征感兴趣，秀丽隐杆线虫是一种居住在土壤中的蛔虫。秀丽隐杆线虫种群中存在雄性和雌雄同体，在形态上存在明显差异。然而，神经系统中性别特异性细胞类型的差异并不容易看到。性别特异性细胞类型的荧光标记已成为识别性别特异性发育缺陷的强大而敏感的工具。已经分离出的突变体在形态上是正常的雄性或雌雄同体，但具有通常只存在于异性身上的神经元。鉴定受这些突变影响的基因及其随后的特征将为性别特异性发育的遗传基础提供新的见解。荧光立体显微镜和成像是必不可少的观察和记录的性缺陷，在体内使用荧光标记检测性别特异性细胞类型。黄实验室对如何建立细胞身份或细胞命运规范感兴趣。MEX-3是秀丽隐杆线虫胚胎发育过程中对细胞命运规范起重要作用的蛋白。MEX-3蛋白最初存在于胚胎的所有细胞中，但在四细胞阶段从后部细胞中耗尽。这种MEX-3分布的变化对于胚胎前后轴的建立是必需的，因此了解MEX-3如何在胚胎中定位是很重要的。初步证据表明，MEX-3的活性降解依赖于两种不同类型的蛋白质修饰，即磷酸化和泛素化。在MEX-3蛋白中鉴定这些修饰的特定位点和鉴定调节这些修饰的基因是非常有趣的。当MEX-3的特定位点发生突变或秀丽隐杆线虫基因组中的其他基因被破坏时，荧光显微镜将能够在体内研究荧光标记的MEX-3的稳定性。科罗拉多学院是美国落基山脉地区唯一一所全国排名的文理学院，招收了来自美国各地和国际的约2000名本科生。生物学系致力于与本科生合作开展原创性研究，荧光显微镜和成像系统每年将为Killian和huang实验室的大约6-10名本科生提供重要的研究和培训机会。此外，生物学课程中的许多课程都以探究性研究实验室体验为特色。有这样经历的学生最有能力应对他们可能面临的任何研究生挑战。该设备将增加和改进与几门课程相关的实验室活动、演示和实验，每年将影响大约200名本科生。成像系统将是一种特别有价值的教学工具，因为它可以使教师和一组学生同时在计算机显示器上观察同一样品。生物系还在学院中发挥着强大的服务作用，提供非专业课程，培养学生对基础生物学研究的强烈欣赏，并使学生能够对许多生物学领域的热门话题（如干细胞研究、基因治疗、转基因生物和环境问题）做出明智的决定和意见。荧光立体显微镜将为非专业学生提供独特而令人兴奋的机会来观察前沿科学。最后，科罗拉多学院积极寻求加强校园的多样性，并提供最先进的仪器，如荧光立体显微镜和成像系统，将有助于吸引代表性不足的少数民族和妇女参与生物学研究。

This Major Research Instrumentation grant supports the acquisition of a fluorescence stereomicroscopeand an imaging system to enhance the research programs of Dr. Darrell J. Killian and Dr. Nancy N.Huang in the Biology Department at Colorado College. The real-time visualization of fluorescentmolecules in living model organisms has become an indispensable technique because it allows the directobservation and documentation of changes in gene activity, the localization of proteins within a cell, andthe tracking of cells and tissues during development. An important question in biology is how changes ingene activity determine sex-specific development. The Killian Lab is interested in the identification andcharacterization of genes that affect sex-specific development in the popular model organismCaenorhabditis elegans (C. elegans), a soil-dwelling roundworm. C. elegans populations contain malesand hermaphrodites, which have some obvious morphological differences. However, sex-specific celltype differences in the nervous system are not easy to see. Fluorescent markers for sex-specific celltypes have emerged as powerful and sensitive tools for the identification of defects in sex-specificdevelopment. Mutants have been isolated that are morphologically normal males or hermaphrodites, butwith neurons that are normally only found in the opposite sex. The identification of the genes affected bythese mutations and their subsequent characterization will provide new insights into the genetic basis ofsex-specific development. Fluorescence stereomicroscopy and imaging are essential for the observationand documentation of sexual defects that are detected in vivo using fluorescent markers for sex-specificcell types. The Huang Lab is interested in how cellular identity, or cell fate specification, is established.MEX-3 is a protein that plays an important role in cell fate specification during C. elegans embryonicdevelopment. The MEX-3 protein is initially present in all cells of the embryo but becomes depleted fromthe posterior cells by the four-cell stage. This change in MEX-3 distribution is required for theestablishment of the anterior/posterior axis of the embryo, thus it is important to understand how MEX-3 islocalized in the embryo. Preliminary evidence suggests that MEX-3 is actively degraded in a manner thatis dependent upon two different types of protein modifications termed phosphorylation and ubiquitination.The identification of the specific sites of these modifications within the MEX-3 protein and theidentification of genes that regulate these modifications are of great interest. Fluorescence microscopywill enable an in vivo study of the stability of fluorescently tagged MEX-3 when specific sites in MEX-3 aremutated or when other genes in the C. elegans genome are disrupted.Colorado College is the only nationally ranked liberal arts college in the Rocky Mountain region of theUnited States and enrolls approximately 2000 undergraduates from all over the United States andinternationally. The Biology Department has a strong commitment to conducting original research withundergraduate collaboration and the fluorescence microscope and imaging system will provide importantresearch and training opportunities for approximately 6-10 undergraduates working in the Killian andHuang labs each year. In addition, many courses in the Biology curriculum feature inquiry-basedresearch lab experiences. Students with such experiences are best equipped to handle any postgraduatechallenges that they might face. The equipment will augment and improve laboratory activities,demonstrations, and experiments associated with several courses and will impact approximately 200undergraduates per year. The imaging system will be a particularly valuable teaching tool because it willenable the instructor and a group of students to simultaneously observe the same sample on a computermonitor. The Biology Department also plays a strong service role in the college by offering non-majorscourses that foster a strong appreciation for basic biology research and empower students to makeinformed decisions and opinions on many popular topics in biology such as stem cell research, genetherapy, genetically modified organisms, and environmental issues. The fluorescence stereomicroscopewill provide non-majors with unique and exciting opportunities to observe cutting-edge science first-hand.Finally, Colorado College actively seeks to enhance diversity on campus and the availability of state-ofthe-art instrumentation, such as a fluorescence stereomicroscope and imaging system, will help attractunderrepresented minorities and women to participate in biology research.