Transgenic Resources for Neuroscience Research

NIMH转基因核心设施有几个主要功能：1）生产用于神经科学研究的转基因研究动物，2）支持神经科学遗传研究相关技术的研究，3）开发新的转基因技术和模型系统，4）参与促进神经科学研究遗传方法的合作项目。 1)生产 过去一年的产量包括： a）通过卵母细胞注射DNA或CRISPR构建体产生的24个转基因小鼠项目，每个项目产生多个品系。 B）5个通过卵母细胞注射产生的转基因大鼠项目，每个项目产生多个品系。 2)技术支持 a）通过冷冻保存生殖细胞或胚胎，已将63个转基因啮齿动物品系存档。 B）通过将携带病原体的动物的品系转移到具有确定的健康状态的动物中，重新衍生了44个品系。 19只绒猴出生。在过去的一年里，随着研究所提出的新研究，增加殖民地的规模至关重要。 d）转基因项目的设计和援助对于缺乏生产转基因动物经验的NIH神经科学实验室仍然很重要。 3)技术发展 核酸酶介导的基因工程：小鼠胚胎发育的独特特征使操纵其基因组成为可能。从早期胚胎中分离出来的小鼠胚胎干细胞（ESC）可以在培养皿中生长，并且仍然保持从单个细胞产生完整小鼠的能力。单个细胞的基因可以被改变，然后扩展并转移到寄养母亲身上。其他物种，包括灵长类动物，对同样的技术很难接受，而这种技术对老鼠很有效。其结果是不断扩大的转基因小鼠品系，科学家们用它们来测试组成小鼠基因组的大约20，000个基因中几乎每一个的作用。 CRISPR/cas9系统可用于更有效地靶向基因，并且与小鼠ESC中的靶向基因一样特异，但也适用于其他物种。核心一直专注于使用CRISPR/cas9来产生条件性敲除基因。这些基因中的重组位点被无害地插入到基因的关键区域的侧翼。只有当重组酶在细胞中表达时，从另一个转基因或瞬时病毒传递的重组酶才被敲除。 也可以对基因进行细微的改变，但进行有条件的修饰需要替换基因的大块。 最初，该核心与NIDA病毒生产核心合作，用标准和CRISPR构建体制备大鼠转基因系。此外，自2016年初以来，核心与国家牙科和颅面研究所（NIDCR）的Nick Ryba合作，通过替换大片段来提高基因修饰的效率。我们已经优化了注射材料的浓度，使用试剂优化供体序列的同源重组，胚胎中的注射位点，以及核酸靶向载体的构建。这些技术现在正被应用于制造小鼠和大鼠转基因，最终目的是使该技术足够有效地用于后代数量较少，妊娠和成熟时间较长的物种，如绒猴。 B）转基因绒猴：在与日本川崎的中央实验动物研究所的Erika Sasaki合作下，该核心已经产生了一种转基因绒猴，该绒猴表达遗传编码的钙指示剂（GECI）和Kusabira橙子，一种荧光标记蛋白。这种转基因猴子的组织在受到特定波长的绿色光的激发时会发出橙子光。为了扩大表达这种基因的动物品系，我们开发了一种人工授精程序，在NIMH群体和约翰霍普金斯大学的王晓琴群体中导致了多胎妊娠。 c）转基因大鼠系：在NIMH转基因核心中产生的大鼠品系已被存放在RRRC（大鼠资源和研究中心），并正在从该组织分发。 与NIDA病毒生产核心合作生产了十多个在特定神经元亚型中表达CRE重组酶的细胞系，该核心表征了表达模式。 两条线被用于神经发生的研究。在一种方法中，表达胸苷激酶（TK）转基因的神经干细胞可以被特异性消融。杰森斯奈德（U不列颠哥伦比亚省）和石楠卡梅隆（NIMH）另一个表达橙子荧光蛋白（OFP）的近交系正在被艾伦科里斯茨基小组（NINDS）用来研究移植神经干细胞的潜力。 e）转基因田鼠 最近，该中心启动了一个制造转基因田鼠的项目。Scott Young（NIMH）和Adam Smith（堪萨斯大学）的这个项目利用CRISPR技术来操纵具有有趣社会行为特征的动物的基因。 （f）支助技术：正在开发若干技术，以提高核心支助职能的能力。冷冻小鼠精子和通过使用更新的方法改进IVF是一项重大努力。冷冻大鼠精子并在可接受的水平下完成大鼠体外受精在所有实验室都是一项具有挑战性的任务。

The NIMH transgenic core facility has several major functions: 1) to produce transgenic research animals for neuroscience research, 2) to support research with associated techniques in genetic research in neuroscience, 3) to develop new transgenic techniques and model systems and 4) to engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Metrics of production over the past year include: a) 24 transgenic mouse projects produced by oocyte injection of DNA or CRISPR constructs, with multiple lines produced for each project. b) 5 transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. 2) Technical Support a) 63 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 44 lines have been re-derived, by transferring lines from pathogen-bearing animals into those with defined health status. c) 19 marmosets have been born. In the last year it has been critical to increase the size of the colony as new research is proposed in the institute. d) Transgenic project design and assistance have continued to be significant to NIH neuroscience labs that lack experience in producing transgenic animals. 3) Technical development a) Nuclease-mediated genetic engineering: There are unique features of a mouses embryonic development that make it possible to manipulate its genome. Mouse embryonic stem cells (ESC) dissected from early embryos can be grown in a dish, and still maintain the ability to generate a complete mouse from a single cell. The genes of that single cell can be altered and then expanded and transferred into a foster mother. Other species, including primates, have been refractory to the same technique that work so well for mice. The result has been an ever expanding collection of transgenic mouse lines that scientists use to test the role of virtually every one of the roughly 20,000 genes that make up a mouse genome. The CRISPR/cas9 system can be used to target genes more efficiently and as specifically as in mouse ESC, but is applicable to other species. The core has been focusing on using CRISPR/cas9 to generate conditional knock out genes. These are genes in which recombination sites are innocuously inserted to flank critical regions of a gene. Only when a recombinase is expressed in the celldelivered from another transgene or a transient virusis the gene knocked out. Subtle changes in the gene can also be made, but making a conditional modification requires the replacement of large chunks of a gene. Initially, the core worked with the NIDA virus production core to make rat transgenic lines with both standard and CRISPR constructs. Moreover, since the beginning of 2016 the core has collaborated with Nick Ryba in the National Institute of Dental and Craniofacial Research (NIDCR) to improve the efficiency of modifying genes by replacing large segments. We have optimized concentration of the injected material, the use of reagents to optimize homologous recombination of donor sequences, the site of injection in embryos, and the construction of nucleic acid targeting vectors. These techniques are now being applied to make mouse and rat transgenics, with the ultimate aim of making the technique efficient enough to use in species with smaller numbers of offspring, and longer gestation and maturation times, such as marmosets. b) Transgenic marmosets: In collaboration with Erika Sasaki at the Central Institute for Experimental Animals in Kawasaki, Japan the core has produced a transgenic marmoset that expresses a genetically encoded calcium indicator (GECI) and Kusabira Orange, a fluorescent marker protein. This transgenic monkeys tissues glow orange when they are excited with a specific wavelength of green light. To expand a line of animals that express this gene we have developed an artificial insemination procedure that has resulted in multiple pregnancies in the NIMH colony and in the Xiaoqin Wangs colony at Johns Hopkins University. c) Transgenic rat lines: Rat lines produced in the NIMH Transgenic core have been deposited in the RRRC (Rat Resource and Research Center) and are being distributed from that organization. Over ten lines that express CRE recombinase in specific neuronal subtypes were produced in collaboration with the NIDA virus production core, which characterized the pattern of expression. Two lines are being used in studies of neurogenesis. In one approach neural stems cells that express the thymidine kinase (TK) transgene can be specifically ablated. Jason Snyder (U British Columbia) and Heather Cameron (NIMH) Another inbred line that expresses the orange fluorescent protein (OFP) is being used by Alan Korestskys group (NINDS) to study the potential of transplanted neural stem cells. e) Transgenic voles Recently, the core initiated a project to make transgenic voles. This project for Scott Young (NIMH) and Adam Smith (University of Kansas) takes advantage of CRISPR technologies to manipulate the genes of an animal with interesting social behavioral traits. f) Support techniques: several techniques are under development to increase the capacity of the core's support functions. Freezing mouse sperm and improving IVF by using newer methods is a major effort. Freezing rat sperm and completing IVF in rats at an acceptable level is a challenging task in all laboratories.