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.

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是否参与阿尔茨海默病的早期突触丧失仍不清楚。C3及其受体CR3也介导小胶质细胞对淀粉样蛋白- β (Abeta)的摄取和降解，这是AD的关键蛋白。因此，升高的C3可能有助于清除有毒的Abeta物种和异常标记神经元突触以进行清除，从而导致AD脑中的突触丢失。C3基因敲除（C3KO）的患者一生都缺乏C3。老年雄性C3KO小鼠海马和皮质突触增多，海马CA3区神经元增多，胶质细胞增多，认知测试表现优于WT小鼠。由于补体介导的突触移除发生在发育过程中，因此很难分析C3缺乏对发育和衰老的影响。因此，我们建议使用诱导型和组成型Cre-loxP系统构建首个补体C3条件敲除小鼠模型（C3 cKO），以评估大脑发育后全局C3KO和生命周期中细胞特异性C3KO对中枢神经系统的影响。Carroll博士，我们的合作者，已经产生了嵌合的floxed C3小鼠，并正在培育它们用于生殖系传播（C3fl/fl），以便它们可以与各种Cre-mouse系杂交，以细胞类型和/或年龄特定的方式消除C3，为许多领域的研究人员创造了一种新的工具。我们假设，大脑发育后的补体c3缺失和骨髓细胞一生中的c3缺失将保护海马中年龄依赖性突触和神经元的损失。因此，我们将建立两种C3 cKO小鼠模型，并比较终身C3缺乏症（C3KO）与脑发育后或髓细胞（仅）C3缺乏症对突触、神经元和胶质细胞的影响。在Aim 1中，我们将产生正常表达C3蛋白、C3- lacz和C3- lacz的絮体补体C3小鼠；ZP-3-Cre老鼠。Carroll博士已经培育出了嵌合的C3和C3- lacz小鼠，并对每一种小鼠进行纯合子生殖系传播。他的实验室将对这些小鼠进行表征，并将C3- lacz小鼠与ZP-3-Cre小鼠杂交，以确定小鼠中C3的表达。在Aim 2中，我们将产生C3诱导条件敲除小鼠，以评估脑发育后启动的全局C3缺失的影响。我们会生成C3fl/fl；UBC-Cre-ERT2+/-小鼠通过将C3fl/fl小鼠从Aim 1杂交到泛素启动子驱动的cre -雌激素受体2小鼠，在P60时给予他莫昔芬，并在4月龄和12月龄时检测海马突触、神经元和胶质细胞。在Aim 3中，我们将组成性敲除髓细胞中的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。我们会生成C3fl/fl；LysMCre+/-小鼠，并在P30、4和12月龄时检测小鼠海马突触、神经元和胶质细胞的变化。这项研究将提供重要的数据，确定C3在大脑布线中的作用，这将最终导致未来对神经退行性疾病模型的研究。