Generation of a Complement C3 Conditional Knockout Mouse

描述(申请人提供)：补体C3有助于大脑发育过程中突触的消除，并在阿尔茨海默病(AD)中升高；但C3是否参与AD的早期突触丢失仍不清楚。C3及其受体CR3还介导小胶质细胞摄取和降解AD中的关键蛋白--淀粉样β蛋白(Abeta)。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供移除，从而导致AD脑中突触的丢失。C3基因敲除(C3KO)在整个生命过程中都缺乏C3。与WT小鼠相比，老龄雄性C3KO小鼠在海马区和皮层有更多的突触，在海马区CA3区有更多的神经元，胶质细胞增多，在认知测试中表现更好。由于补体介导的突触移除发生在发育过程中，很难分析C3缺乏对发育和衰老的影响。因此，我们建议使用可诱导的和结构性的Cre-loxP系统来建立第一个补体C3条件基因敲除小鼠模型(C3CKO)，以评估脑发育后全局C3KO和细胞特异性C3KO在整个生命过程中的中枢神经系统效应。我们的合作者Carroll博士已经培育了嵌合的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小鼠品系杂交，以特定细胞类型和/或年龄的方式消除C3，为许多领域的研究人员提供了一种新的工具。我们假设，脑发育后的整体补体C3缺失和终生髓系细胞中的C3缺失将对年龄依赖性突触和海马神经元丢失具有保护作用。因此，我们将建立两种C3CKO小鼠模型，比较终生C3缺乏症(C3KO)与脑发育后C3缺乏症或仅在髓系细胞中(仅)对突触、神经元和胶质细胞的影响。在目标1中，我们将产生正常表达C3蛋白的补体C3小鼠，C3-LacZ和C3-LacZ；ZP-3-Cre小鼠。卡罗尔博士已经培育出嵌合的C3和C3-LacZ小鼠，并正在培育每一种纯合子生殖系传播的品系。他的实验室将对这些小鼠进行鉴定，并将C3-LacZ小鼠与ZP-3-Cre小鼠杂交，以确定C3在小鼠中的表达。在目标2中，我们将产生C3可诱导的条件性基因敲除小鼠，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，得到C3fl/fl；Ubc-Cre-ERT2+/-小鼠，在P60用他莫昔芬治疗，并在4个月和12个月龄时检测海马区突触、神经元和胶质细胞。在目标3中，我们将结构性地敲除髓系细胞中的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C3fl/fl；LysMCre+/-小鼠，并在P30、4个月和12个月龄时检查小鼠海马区突触、神经元和胶质细胞的变化。这项研究将提供确定C3在大脑连接中的作用的重要数据，这最终将导致未来对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.