PDF(Pubmed)
Abstract：
We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability.
摘要：
我们开发了一种方法，该方法利用核包膜的荧光标记以及细胞计数分选来选择性分离浦肯野细胞（PC）核。从SUN1报告小鼠开始，我们用GFP标记的包膜来确认PC细胞核可以与其他细胞类型准确分离。然后，我们开发了一种基于抗体的方案，以使PC核分离更加强大，并且适用于任何基因型背景的小脑组织。核膜蛋白RanBP2的免疫荧光标记能够从C57BL/6小脑中分离PC核。通过分析PC标记的表达，核大小,和核仁数,我们证实了我们的方法提供了PC核的纯分数。为了证明其适用性，我们从脊髓小脑共济失调7型(SCA7)小鼠中分离出PC核,并鉴定了已知和新的疾病相关基因的转录变化.访问纯PC核提供了对PC生物学和病理学的见解，包括选择性神经元脆弱性的性质。
