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19个转基因小鼠项目，每个项目产生多个品系。 B）已经开展了五个利用胚胎干细胞的项目。 c）通过卵母细胞注射产生的三个转基因大鼠项目，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，已存档了87个转基因啮齿动物品系。 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39个品系。 c）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（CRISPR）：小鼠胚胎发育已经被观察和操纵了几十年。基因操作利用了在培养皿中培养全能细胞（胚胎干细胞，ESC）的能力。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。 现在，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。核心重点是使用CRISPR/Cas9在小鼠、大鼠和灵长类动物中产生条件性敲除基因。在这些动物中，重组信号被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，基因功能才被破坏。也可以对基因进行细微的改变，但进行条件性修饰会取代基因的大片段。 去年，该核心受益于与美国国家牙科和颅面研究所（NIDCR）的Nick Ryba的合作，通过替换大片段来提高修饰基因的效率。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，核心已经产生了一系列转基因绒猴，其表达遗传编码的钙指示剂（GECI），这将允许体内神经元活动的可视化。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实现了多胎妊娠。这些动物被用于感觉皮层活动的实验。 c）转基因大鼠系：来自世界各地的50多个实验室已经要求转基因核心与NIDA实验室合作生产的转基因大鼠品系。这些线路由RRRC（大鼠资源和研究中心）维护和分发。在特定的神经元亚型中表达CRE重组酶的这些细胞系中有十多个正在继续表征其表达模式。 一种表达橙子荧光蛋白（OFP）的近交系大鼠正被艾伦·科里斯茨基的研究小组（NINDS）用于研究移植神经干细胞的潜力。 （d）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。在所有实验室中，冷冻大鼠精子并在可接受的水平上实现大鼠体外受精是一项具有挑战性的任务。一种通过外周循环将腺相关病毒（AAV）引入大脑的方法也在进行中。这种方法将允许更广泛地递送转基因，并且消除通过手术干预进行颅内递送的需要。

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) 19 transgenic mouse projects produced by oocyte injection of DNA or CRISPR constructs, with multiple lines produced for each project. b) Five projects using embryonic stem cells (ESC) have been undertaken. c) Three transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. 2) Technical Support a) 87 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 39 lines have been re-derived, by transferring lines from pathogen-bearing animals into those with defined health status. c) 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 (CRISPR): Mouse embryonic development has been observed and manipulated for decades. Genetic manipulation has taken advantage of the ability to grow totipotent cells (embryonic stem cells, ESC) in a dish. Other species, including primates, have been refractory to the same technique that works 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. Now, 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 focused on using CRISPR/Cas9 to generate conditional knock out genes in mice, rats and primates. In these animals, recombination signals are inserted to flank critical regions of a gene. Only when a recombinase is expressed in the cell is the gene function disrupted. Subtle changes in the gene can also be made, but making a conditional modification replaces large segments of a gene. The core has over the last year benefited from a collaboration 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 line of transgenic marmosets that expresses a genetically encoded calcium indicator (GECI) which will allow the visualization of neuronal activity in vivo. 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 Wang's colony at Johns Hopkins University. These animals are being used for experiments on the activity in the sensory cortex. c) Transgenic rat lines: Over 50 laboratories from around the world have requested transgenic rat lines that were produced by the transgenic core in collaboration with labs in NIDA. These lines are being maintained and distributed by the RRRC (Rat Resource and Research Center). More than ten of these lines that express CRE recombinase in specific neuronal subtypes are continuing to be characterized for their patterns of expression. An inbred line of rats that expresses the orange fluorescent protein (OFP) is being used by Alan Korestsky's group (NINDS) to study the potential of transplanted neural stem cells. d) 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 achieving IVF in rats at an acceptable level is a challenging task in all laboratories. A method of introducing adeno-associated virus (AAV) to the brain through the peripheral circulation is also underway. This method would allow more widespread delivery of a transgene and eliminate the need for intracranial delivery by surgical intervention.