Generation of a Complement C3 Conditional Knockout Mouse

描述（由申请人提供）：补体C3有助于脑发育期间的突触消除，并且在阿尔茨海默病（AD）中升高;但C3是否参与AD中的早期突触丧失仍不清楚。C3及其受体CR 3还介导小胶质细胞对淀粉样蛋白β（A β）的摄取和降解，A β是AD中的关键蛋白。因此，升高的C3可能有助于毒性Abeta物质的去除和神经元突触的异常标记以去除，导致AD脑中的突触丢失。C3敲除（C3 KO）是终身C3缺乏。与WT小鼠相比，老年雄性C3 KO小鼠在海马和皮质中具有更多的突触，海马CA 3中的神经元，增加的胶质增生，并且在认知测试中表现更好。由于补体介导的突触清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系统产生第一个补体C3条件性敲除小鼠模型（C3 cKO），以评估大脑发育后全局C3 KO和细胞特异性C3 KO在整个生命周期中的CNS效应。我们的合作者卡罗尔博士已经产生了嵌合体floxed C3小鼠，并正在为生殖系传递（C3 fl/fl）进行繁殖，以便它们可以与各种Cre-mouse系杂交，以细胞类型和/或年龄特异性方式消除C3，为许多领域的研究人员提供新的工具。我们假设，全球补体C3缺失脑发育后和C3缺失髓细胞通过生活将保护对年龄依赖性突触和海马神经元的损失。因此，我们将开发两种C3 cKO小鼠模型，并比较终身C3缺陷（C3 KO）与脑发育后或髓样细胞（仅）中C3缺陷对突触、神经元和神经胶质的影响。在目标1中，我们将产生通常表达C3蛋白的floxed补体C3小鼠，C3-LacZ和C3-LacZ;ZP-3-Cre小鼠。卡罗尔博士已经产生了嵌合体floxed C3和C3-LacZ小鼠，并正在繁殖纯合子种系传播的每一个线。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。在目标2中，我们将产生C3诱导的条件性敲除小鼠，以评估脑发育后启动的全局C3缺失的影响。我们将生成C3 fl/fl;通过将来自Aim 1的C3 fl/fl小鼠与泛素启动子驱动的Cre-雌激素受体2小鼠杂交，在P60用他莫昔芬处理小鼠，并在4月龄和12月龄检查海马中的突触、神经元和神经胶质。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。我们将产生C3 fl/fl;LysMCre+/-小鼠，并在P30、4月龄和12月龄检查小鼠海马中突触、神经元和神经胶质的变化。这项研究将提供重要的数据，确定C3在大脑布线中的作用，这将最终导致未来的神经退行性疾病模型的研究。

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.