Cytoskeletal Function in C. elegans Embryos

我们要求资金购买两个高容量和多功能的BioRad热力自行车。 它们将取代目前单一的更老、更低容量的热循环器 由我的实验室和我附近的同事和教职员工的相邻实验室共享 克里斯·多伊和戴安娜·利布达。目前，由于我们的机器的年龄和它的有限 由于缺乏能力，以及缺乏其他体制选择，我们遭受了缺乏准入的痛苦 为了扩增构建转基因菌株，我们的研究需要。这个 CRISPR/Cas9的发现及其在基因组编辑中的应用带来了革命性的变化 所有系统中的分子遗传学，包括线虫和我们有限的热循环器 能力现在经常限制和减慢我们的进展。通过购买两个新的 容量更高的机器，并与另外两个实验室共享安排，我们将 不仅在我自己的实验室中解除访问限制，还在我的两个实验室中解除访问限制 最亲密和身体相邻的同事。在高分辨率方面的显著进展 光学显微镜使我们能够产生与内源性荧光蛋白融合的 LOGI越来越强大，我们也在实施更高度机械化 结构/功能研究，需要建立转基因株系，以表达 突变的转基因以取代内源基因。最后，我们现在也开始 利用光遗传学更严格地探索基因需求。我们需要增加 热循环器产生转基因构建物的能力，用于所有这些强大的 分子遗传学方法。

We request funds to purchase two high capacity and versatile BioRad Thermal Cyclers. These will replace a single much older and lower capacity thermal cycler currently shared by my lab and the adjacent labs of my nearby colleagues and faculty members Chris Doe and Diana Libuda. Currently, due to the age of our machine and its limited capacity, and a lack of other institutional options, we have suffered from lack of access to amplification for constructing transgenic strains we need for our research. The discovery of CRISPR/Cas9 and its applications to genome editing have revolutionized molecular genetics in all systems, including C. elegans, and our limited thermal cycler capacity is now frequently limiting and slowing our progress. By purchasing two new higher capacity machines, and having a shared arrangement with two other labs, we will relieve not only the limitation to access in my own lab but also in the labs of two of my closest and physically adjacent colleagues. The remarkable advances in high resolution light microscopy make our ability to generate fluorescent protein fusions to endogenous loci more and more powerful, and we also are implementing more highly mechanistic structure/function studies that require the creation of transgenic lines that express mutant transgenes to replace endogenous genes. Finally, we also are now beginning to use optogenetics to more rigorously explore gene requirements. We need increased thermal cycler capacity to generate transgene constructs for use in all of these powerful molecular genetic approaches.