Generation of a Complement C3 Conditional Knockout Mouse

补体 C3 条件性敲除小鼠的生成

基本信息

批准号：
8741912
负责人：
CYNTHIA A LEMERE
金额：
$ 20.71万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8741912
关键词：
ARHGEF5 gene Age Aging Alzheimer&apos s Disease Amyloid Amyloid beta-Protein Brain Brain region Breeding C3 Deficiency Cells Chemicals Cognitive Complement Complement 3d Crossbreeding Data Development Disease Disease model Enzyme-Linked Immunosorbent Assay Estrogen Receptor 2 Event Excision Excitatory Synapse Future Gender Generations Germ Lines Gliosis Health Hippocampus (Brain)Immune Immunofluorescence Immunologic Immunohistochemistry In Situ Hybridization Inhibitory Synapse Injection of therapeutic agent Knock-out Knockout Mice Label LacZ Genes Lead Life Liver Longevity Macrophage-1 Antigen Measurement Mediating Microglia Modeling Mus Myeloid Cells Natural Immunity Nerve Degeneration Neurodegenerative Disorders Neuroglia Neurons Outcome Measure Patients Performance Phenotype Play Proteins Research Personnel Resolution Role Serum Synapses System Tamoxifen Testing Ubiquitin Western Blotting Work age effect age related aged aging brain cell type critical period male mild cognitive impairment mouse development mouse model neuron loss novel promoter public health relevance ranpirnase tool transmission process uptake

项目摘要

DESCRIPTION (provided by applicant): Complement C3 contributes to synaptic elimination during brain development and is elevated in Alzheimer's disease (AD); but whether C3 is involved in early synaptic loss in AD remains unknown. C3 and its receptor CR3 also mediate microglial uptake and degradation of amyloid-beta (Abeta), a key protein in AD. Thus, elevated C3 may contribute to both the removal of toxic Abeta species and the aberrant tagging of neuronal synapses for removal, resulting in synaptic loss in AD brain. C3 knockout (C3KO) are C3-deficient through life. Aged male C3KO mice have more synapses in hippocampus and cortex, neurons in CA3 of hippocampus, increased gliosis, and perform better in cognitive tests compared to WT mice. Because complement-mediated synaptic removal takes place during development, it is difficult to dissect the developmental versus aging effects of C3- deficiency. Therefore, we propose to generate the first complement C3 conditional knockout mouse models (C3 cKO) using inducible and constitutive Cre-loxP systems that to assess the CNS effects of global C3KO after brain development and cell-specific C3KO throughout life. Dr. Carroll, our collaborator, has generated chimeric floxed C3 mice and is breeding them for germ-line transmission (C3fl/fl) so that they can be crossed with various Cre-mouse lines to eliminate C3 in a cell type- and/or age-specific manner, generating a novel tool for researchers in many fields. We hypothesize that global complement C3-deletion after brain development and C3-deletion in myeloid cells through life will be protective against age-dependent synapse and neuron loss in hippocampus. Therefore, we will develop two C3 cKO mouse models and compare the effects of lifelong C3-deficiency (C3KO) with C3-deficiency after brain development or in myeloid cells (only) on synapses, neurons, and glia. In Aim 1, we will generate floxed complement C3 mice that normally express C3 protein, C3-LacZ and C3-LacZ;ZP-3-Cre mice. Dr. Carroll has generated chimeric floxed C3 and C3-LacZ mice and is breeding each line for homozygous germline transmission. His lab will characterize these mice and cross the C3-LacZ mice with ZP-3-Cre mice to determine C3 expression in mice. In Aim 2, we will generate C3 inducible conditional knockout mice to assess the effects of global C3 deletion initiated after brain development. We will generate C3fl/fl;UBC-Cre-ERT2+/- mice by crossing the C3fl/fl mice from Aim 1 to ubiquitin-promoter driven Cre-Estrogen receptor 2 mice, treat the mice with tamoxifen at P60, and examine synapses, neurons and glia in hippocampus at 4 mo and 12 mo of age. In Aim 3, we will constitutively knockout C3 in myeloid cells to determine whether C3 produced by these immune cells contributes to synaptic pruning and neuronal health. We will generate C3fl/fl;LysMCre+/- mice and examine mice for changes in synapses, neurons and glia in hippocampus at P30, 4 mo and 12 mo of age. This study will provide important data determining the role of C3 on brain wiring, which will lead ultimately to future studies in models of neurodegenerative diseases.

描述（申请人提供）：补体C3在大脑发育过程中有助于消除突触，并且在阿尔茨海默氏病（AD）中升高；但是，C3是否参与AD的早期突触损失仍然未知。 C3及其受体CR3还介导淀粉样蛋白β（ABETA）的小胶质细胞摄取和降解，这是AD中的关键蛋白质。因此，升高的C3可能导致去除有毒Abeta物种和神经元突触的异常标记以去除，从而导致AD脑的突触损失。 C3敲除（C3KO）在生活中缺乏C3。老年雄性C3KO小鼠在海马和皮质中具有更多的突触，与WT小鼠相比，在海马CA3中的神经元，海马的神经元增加，在认知测试中表现更好。由于在发育过程中进行了补体介导的突触去除，因此很难剖析C3缺乏症的发育效果与衰老效应。因此，我们建议使用诱导性和组成型CRE-LoxP系统生成第一个补体C3条件基因敲除小鼠模型（C3 CKO），以评估脑发育后全局C3KO的CNS效应，并且在整个生命中都可以评估细胞特异性C3KO。我们的合作者Carroll博士已经生成了嵌合的Floxed C3小鼠，并将它们繁殖用于种系传输（C3FL/FL），以便它们可以与各种Cre-Mouse系列交叉，以以细胞类型和/或年龄为特定的方式消除C3，从而为许多领域的新颖工具生成一个新颖的工具。我们假设在脑发育后的全球补体C3-局部和髓样细胞中的C3缺失通过生命将对海马中的年龄依赖性突触和神经元丧失进行保护。因此，我们将开发两种C3 CKO小鼠模型，并比较终身C3缺乏症（C3KO）与脑发育后或在髓样细胞中（仅）对突触，神经元和神经胶质的影响。在AIM 1中，我们将生成通常表达C3蛋白，C3-LacZ和C3-LacZ; ZP-3-CRE小鼠的Floxed Reploxement C3小鼠。 Carroll博士已经产生了嵌合的C3和C3-Lacz小鼠，并正在繁殖每条线以进行纯合种系传输。他的实验室将以ZP-3-CRE小鼠的身份表征这些小鼠，并越过C3-Lacz小鼠，以确定小鼠中的C3表达。在AIM 2中，我们将生成C3诱导的条件敲除小鼠，以评估脑发育后启动的全局C3缺失的影响。我们将通过将C3FL/FL小鼠从AIM 1跨到Ubiquitin促进蛋白驱动的Cre-strogen受体2小鼠2小鼠来生成C3fl/fl; UBC-CRE-ERT2 +/-小鼠，在P60处用他莫昔芬治疗小鼠，并检查Hippocampus在Hippocampus，Hippocampus在Hippocampus，Hippocampus在4 mo and Mo and Mo and 12 Mo and age and 12 Mo age。在AIM 3中，我们将组成性地敲除髓样细胞中的C3，以确定这些免疫细胞产生的C3是否有助于突触修剪和神经元健康。我们将生成C3FL/FL; lysmcre +/-小鼠，并检查小鼠在P30，4个月和12个月中的海马中突触，神经元和神经胶质的变化。这项研究将提供重要的数据，以确定C3在脑接线上的作用，这最终将导致未来在神经退行性疾病模型中的研究。