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Transgenic Resources for Neuroscience Research

神经科学研究的转基因资源

基本信息

批准号：
10706211
负责人：
James Pickel
金额：
$ 210.5万
依托单位：
NATIONAL INSTITUTE OF MENTAL HEALTH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10706211
关键词：
2019-nCoV ACE2 Animal Experimentation Animal Model Animals Behavior Binding Binding Sites Biochemical Blood - brain barrier anatomy Blood Circulation Brain Callithrix Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complex Core Facility Cryopreservation Development Disease Drug Delivery Systems Electrophysiology (science)Embryo Exons Fibroblasts Foxes Frequencies Functional disorder Gene Expression Gene Transfer Genes Genetic study Genome Goals Home Human Infant Institution International Intravenous Laboratories Link Liver Mental disorders Methods Monkeys National Institute of Mental Health Nervous System Physiology Neurosciences Research Patients Peripheral Primates Production Proteins Rattus Reproducibility Research Research Personnel Resources Rodent Role Science Symptoms System Techniques Tertiary Protein Structure Therapeutic Transgenes Transgenic Animals Transgenic Mice Transgenic Model Transgenic Organisms United States National Institutes of Health Variant Viral Virus Virus Diseases Work adeno-associated viral vector brain cell brain research design genetic manipulation in vitro Model innovation neural new technology vector

项目摘要

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

1)啮齿动物转基因生产