Generation of a Complement C3 Conditional Knockout Mouse

补体 C3 条件性敲除小鼠的生成

• 批准号: 8638529
• 负责人: CYNTHIA A LEMERE
• 金额: $ 26.82万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638529
• 关键词: ARHGEF5 gene; Age; Aging; Alzheimer' 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

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（由申请人提供）：补体 C3 有助于大脑发育过程中的突触消除，并在阿尔茨海默病 (AD) 中升高；但 C3 是否参与 AD 早期突触丧失仍不清楚。 C3 及其受体 CR3 还介导小胶质细胞摄取和降解淀粉样蛋白 - β (Abeta)，这是 AD 的关键蛋白。因此，升高的 C3 可能有助于去除有毒的 Abeta 物质，并导致神经元突触的异常标记以去除，从而导致 AD 大脑中的突触损失。 C3 基因敲除 (C3KO) 患者终生都缺乏 C3。与WT小鼠相比，老年雄性C3KO小鼠的海马和皮层突触更多，海马CA3神经元更多，神经胶质增生增加，并且在认知测试中表现更好。由于补体介导的突触去除发生在发育过程中，因此很难剖析 C3 缺乏对发育与衰老的影响。因此，我们建议使用诱导型和组成型 Cre-loxP 系统生成第一个补体 C3 条件敲除小鼠模型 (C3 cKO)，以评估大脑发育后整体 C3KO 和整个生命周期中细胞特异性 C3KO 的 CNS 影响。我们的合作者 Carroll 博士已经培育出了嵌合 floxed C3 小鼠，并正在培育它们进行种系传递 (C3fl/fl)，以便它们可以与各种 Cre 小鼠品系杂交，以细胞类型和/或年龄特异性的方式消除 C3，为许多领域的研究人员提供了一种新工具。我们假设大脑发育后的全局补体 C3 缺失和一生中骨髓细胞中的 C3 缺失将能够防止海马中年龄依赖性突触和神经元损失。因此，我们将开发两种 C3 cKO 小鼠模型，并比较终身 C3 缺乏 (C3KO) 与大脑发育后或仅骨髓细胞中 C3 缺乏对突触、神经元和胶质细胞的影响。在目标 1 中，我们将生成正常表达 C3 蛋白的 floxed 补体 C3 小鼠、C3-LacZ 和 C3-LacZ；ZP-3-Cre 小鼠。 Carroll 博士已经培育出嵌合 floxed C3 和 C3-LacZ 小鼠，并正在培育每个品系以实现纯合种系传播。他的实验室将表征这些小鼠，并将 C3-LacZ 小鼠与 ZP-3-Cre 小鼠杂交，以确定小鼠中的 C3 表达。在目标 2 中，我们将生成 C3 诱导条件敲除小鼠，以评估大脑发育后启动的全局 C3 缺失的影响。我们将通过将 Aim 1 的 C3fl/fl 小鼠与泛素启动子驱动的 Cre-雌激素受体 2 小鼠杂交来产生 C3fl/fl;UBC-Cre-ERT2+/- 小鼠，在 P60 时用他莫昔芬治疗小鼠，并在 4 个月和 12 个月龄时检查海马中的突触、神经元和神经胶质细胞。在目标 3 中，我们将组成性敲除骨髓细胞中的 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，并在 P30、4 个月和 12 个月时检查小鼠海马突触、神经元和胶质细胞的变化。这项研究将提供重要的数据来确定 C3 在大脑布线中的作用，这最终将导致神经退行性疾病模型的未来研究。