Transgenic Resources for Neuroscience Research

1)生产啮齿动物转基因产品 小鼠转基因生产项目：22个 大鼠转基因生产项目：4 冷冻保存项目：26个 重新衍生项目：20个 2)SARS-Cov2 A)成纤维细胞培养已经使用实验室开发的方法(见下文)进行了几年的繁殖。这些培养物已被证明是ACE2表达的体外模型。ACE2蛋白(作为SARS-CoV2病毒刺突蛋白的结合部位)已被导入培养的绒猴成纤维细胞。在这些成纤维细胞上表达了绒猴蛋白和人类蛋白，以比较病毒感染的可能性。嵌合的或人源化的绒猴ACE2也在成纤维细胞中表达。表达这三种结构的几个细胞系已经被永生化，以创建可重复的试验床，使其他研究人员能够评估分离的病毒刺突蛋白的结合。 B)在培养中生长的Marmoset胚胎被注入了改变ACE2基因的CRISPR构建体。病毒刺突蛋白的结合需要ACE2蛋白结构域。在培养的绒猴胚胎中，这些结构域中的两个已经改变。编码这些结构域的外显子片段的靶向频率正在优化。 3)猕猴转基因生产技术 对于大多数研究来说，操纵胚胎来生产一系列转基因绒猴不再是一个可行的选择。我们已经开发出一种使用AAV载体变体将转基因引入脑细胞的更有效的方法。这些独特的AAVs可以穿过血脑屏障并迁移到大脑中，可以输入到外周循环中。它们避免像大多数病毒那样在肝脏积聚，而是优先进入大脑。这些变种是与加州理工大学格拉迪纳鲁实验室合作开发的；然后通过核心设施中潜在变种的筛选池来选择变种。我们已经培育了几种携带转基因并在大脑中表达的动物。与其他合作者一起，我们鉴定了这两个具有更高转导频率的新变体，并设计了载体有效载荷，以在特定细胞中表达外源基因，并使用修改后的CRISPR分子控制内源基因的表达。 东半球灵长类动物的AAV变异是这项工作的重要延伸： Chuapoco M，Flytzanis N，Goeden N，Octeau JC，Roxas Km，Chan KY，Scherrer J，Winchester J，Blackburn RJ，Campos LJ，Arokiaraj CM，Miles Tf，Jang MJ，Vendymatti J，Deverman Be，Pickel J，Fox As，Gradinaru V.使用AAV通过婴儿脑内进行基因转移。 BioRxiv 2022.01.08.475342；doi：https://doi.org/10.1101/2022.01.08.475342

1) Production of rodent transgenics Mouse transgenic production projects: 22 Rat transgenic production projects: 4 Cryopreservation projects: 26 Rederivation projects: 20 2) SARS-Cov2 a) Fibroblast cultures have been propagated using methods developed in the lab (see below) over several years. These cultures have proven useful as in vitro models of ACE2 expression. The ACE2 protein (which acts as a binding site for the SARS-CoV2 virus spike protein) has been introduced into cultured marmoset fibroblasts. Both the marmoset protein and the human protein have been expressed on these fibroblasts to compare the potential for viral infection. Chimeric, or humanized marmoset ACE2 has also been expressed in fibroblasts. Several cell lines expressing each of these three constructs have been immortalized to create reproducible testbeds to enable other investigators to evaluate the binding of isolated viral spike protein. b) Marmoset embryos grown in culture have been injected with CRISPR constructs that alter the ACE2 gene. Binding of the viral spike protein requires ACE2 protein domains. Two of these domains have been altered in cultured marmoset embryos. The frequency of targeting of the segments of the exons that encode these domains is being optimized. 3) Techniques for transgenic production in marmoset monkeys Manipulating embryos to produce a line of transgenic marmosets is no longer a feasible option for most research. We have developed a more effective method of introducing transgenes into brain cells using AAV vector variants. These unique AAVs, which can cross the blood-brain barrier and migrate into the brain, can be infused into the peripheral circulation. They avoid accumulating in the liver like most virus, but instead preferentially home to the brain. These variants were developed in collaboration with the Gradinaru laboratory at Cal Tech; the variants were then selected by screen pools of potential variants in the core facility. We have produced several animals that carry transgenes and express them in the brain. With other collaborators we have characterized both new variants with higher transduction frequency and have designed vector payloads to express the exogenous gene in specific cells as well as control endogenous gene expression using modified CRISPR molecules. AAV variant development to Old World primates has been an important extension of this work: Chuapoco M, Flytzanis N, Goeden N, Octeau JC, Roxas KM, Chan KY, Scherrer J, Winchester J, Blackburn RJ, Campos LJ, Arokiaraj CM, Miles TF, Jang MJ, Vendemiatti J, Deverman BE, Pickel J, Fox AS, Gradinaru V. Intravenous gene transfer throughout the brain of infant Old World primate using AAV. bioRxiv 2022.01.08.475342; doi: https://doi.org/10.1101/2022.01.08.475342