Transgenic Resources for Neuroscience Research

NIMH转基因核心设施有几个主要功能：1）为神经科学研究生产转基因研究动物，2）开发新的转基因技术和模型系统，3）参与促进神经科学研究遗传方法的合作项目。 1)生产 过去一年的生产规模包括： a）由CRISPR构建体产生的12个转基因啮齿动物项目，其中每个项目在大鼠或小鼠中产生多个品系。 B）没有使用标准质粒构建体的项目，和 c）一个开发三种基因独特的胚胎干细胞系的项目。 2)技术支持 a）通过冷冻保存生殖细胞或胚胎，已将56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25个品系。 （c）使用独特的外科技术完成了2个项目。 3)技术发展 a）今年，在冠状病毒病（COVID-19）大流行期间，由于大多数研究人员远离他们通常的实验室和动物群，因此保存有价值的研究动物模型变得至关重要。如果没有一个可预测的时间，当研究人员可以返回物理工作场所，有失去的危险线，采取了广泛的资源来开发。因此，核心设施在此期间一直在工作，以便首先通过冷冻保存保存品系，然后恢复品系用于重新研究。对五十六株系进行了超低温保存。在本财政年度结束之前，至少有10条线将被重新推导，并准备在研究重新开始时进行实验。 B）在过去的一年里，核心已经使用CRISPR技术为神经科学研究产生了大多数转基因啮齿动物。已经使用优化的CRISPR引导物和同源臂将启动子和编码序列插入到转录中性基因组位点（例如小鼠和大鼠的ROSA基因座）中来产生一些转基因。通过敲入基因的大区域以在编码外显子的侧翼添加重组信号以产生条件性敲除，已经产生了其他转基因。另一组转基因将CRE或FLP重组酶添加到现有的转录物中，以保持来自内源性转录启动子的表达的保真度。核心细胞很少使用质粒或ESC（胚胎干细胞）介导的转基因方法产生转基因。 c）通过操纵胚胎产生一系列生殖系转基因动物来产生转基因绒猴的过程对大多数研究来说不再是一个可行的选择。这些方法可用于产生表达重组酶的转基因株系。然而，已经开发了使用AAV载体引入转基因的更有效的方法，所述AAV载体穿过血脑屏障并在脑中的神经元中表达转基因。通过与加州理工学院的Gradinaru实验室合作，我们已经培育出了几种携带转基因并在大脑中表达的动物。目前正在完成进一步的工作，以便在校内方案中使用这项技术。 d）ACE 2蛋白在细胞表面表达，并被SARS-CoV 2病毒的受体结合结构域（RBD）靶向。这使得病毒进入细胞并产生生产性感染。研究动物对这种病毒有一系列的易感性。修改动物ACE 2基因以匹配人类基因可能会推进对COVID-19的研究，特别是对人们知之甚少的神经学特征的研究。这种修饰的基因已被引入培养系统（细胞和动物受精卵），以测试这种方法。 e）与NIDA的研究人员合作产生了一组表达CRE重组酶的大鼠。这些线路继续通过老鼠资源和研究中心分发。其他表达CRE的细胞系一旦被鉴定，将通过RRRC进行分配。 f）表达绿色荧光蛋白的小鼠ESC（胚胎干细胞）系继续获得许可。

The NIMH transgenic core facility has several major functions: 1) to produce transgenic research animals for neuroscience research, 2) to develop new transgenic techniques and model systems, and 3) to engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Metrics of production over the past year include projects: a) 12 transgenic rodent projects produced by CRISPR constructs, with multiple lines produced for each project in rats or mice. b) no projects that use standard plasmid constructs, and c) one project developing three, genetically unique embryonic stem cell lines. 2) Technical Support a) 56 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 25 lines have been re-derived, by transferring lines from pathogen-bearing animals into those with defined health status. c) 2 projects were completed using unique surgical techniques. 3) Technical development a) In this year, during the Coronavirus Disease (COVID-19) pandemic, as most researchers worked away from their usual laboratories and animal colonies it became critical to preserve valuable research animal models. Without a predictable time when researchers could return to the physical workplace, there was the danger of losing lines that had taken extensive resources to develop. For that reason, the core facility worked through this period in order to first preserve lines by cryopreservation and later to recover lines for renewed research. Fifty-six lines were cryopreserved. Before the end of the fiscal year, at least 10 lines will be rederived and ready for experiments that will follow when research begins anew. b) Over the last year the core has generated most transgenic rodents for neuroscience research using CRISPR technology. Some transgenics have been generated using optimized CRISPR guides and homology arms to insert promoters and coding sequence into transcriptionally neutral genomic sites, such as the ROSA locus of mice and rats. Other transgenics have been produced by knocking in large regions of a gene to add recombination signals flanking coding exons to generate conditional knockouts. Another set of transgenics add CRE or FLP recombinases to existing transcripts in order to maintain the fidelity of expression from endogenous transcriptional promoters. Only rarely does the core produce transgenics using plasmid- or ESC (embryonic stem cell)-mediated transgenic methods. c) The process of generating transgenic marmosets by manipulating embryos to produce a line of germline transgenic animals is no longer a feasible option for most research. These methods are useful for producing lines of transgenics that express recombinases. However, a more effective method of introducing a transgene using AAV vectors that crosses the blood brain barrier and expresses a transgene in neurons in the brain has been developed. In collaboration with the Gradinaru laboratory at Cal Tech, we have produced several animals that carry transgenes and express them in the brain. Further work is being completed to use this technology in the intramural program. d) The ACE2 protein is expressed on the surface of cells and is targeted by the receptor binding domain (RBD) of the SARS-CoV2 virus. This allows the virus to enter the cell and create a productive infection. Research animals have a range of susceptibility to this virus. Modifying an animals ACE2 gene to match the human gene may advance the study of COVID-19, especially the neurological features that are poorly understood. This modified gene has been introduced in culture systems (cells and animal zygotes) to test this method. e) A panel of rats that express the CRE recombinase was generated in a collaboration with investigators at NIDA. Those lines continue to be distributed through the Rat Resource and Research Center. Other CRE-expressing lines will be distributed through RRRC once they are characterized. f) Mouse ESC (embryonic stem cell) lines that express the green fluorescent protein continue to be licensed.