Human chromosome isolation from short-term lymphocyte culture for flow cytometry
Human chromosome isolation from short-term lymphocyte culture for flow cytometry
复制标题
从短期淋巴细胞培养物中分离人类染色体用于流式细胞术
作者：
Loh‐Chung Yu;J. Aten;J. Gray;A. Carrano
流式细胞术特别适合于区分不同dna含量1，2或dna碱基组成3，4的分离的哺乳动物染色体。用这种技术，用Hoechst 33258染色，可将人类24种类型的染色体分成15组。用Hoechst 33258(HO)和显色霉素A3(CA3)，用双光束流式细胞术3，6可以区分20组人类染色体，并估计每组染色体的相对频率。Flow测量提供了对染色体重排2、8和频率变化敏感的细胞群体(流动核型7)的平均染色体组合的精确描述；也使单个染色体能够被纯化，例如用于基因图谱9。到目前为止，只从已建立的成纤维细胞培养中分离出染色体用于流式细胞术。然而，这种培养的维护既耗时又昂贵，而且在延长的成纤维细胞培养过程中可能会发生染色体重排的风险。在这里，我们描述了两种从短期淋巴细胞培养中分离中期染色体的方法，这些中期染色体在形态上是完整的，适合于流式细胞仪分析和纯化，并且具有高相对分子质量的DNA。
Flow cytometry is uniquely suited for distinguishing between isolated mammalian chromosomes differing in DNA content1,2 or DNA-base composition3,4. Using this technique, the 24 types of human chromosomes can be resolved into 15 groups when stained with Hoechst 33258 (refs 3, 5). When Hoechst 33258 (HO) and chromomycin A3 (CA3) are used, 20 groups of human chromosomes can be distinguished by dual-beam flow cytometry3,6, and the relative frequency of chromosomes in each group estimated. Flow measurements provide a precise description of the average chromosome complement of the cell population (a flow karyotype7) that is sensitive to chromosomal rearrangements2,8 and frequency changes; and also enable individual chromosomes to be purified, for example, for gene mapping9. Until now, chromosomes have been isolated for flow cytometry only from established fibroblast cultures. However, the maintenance of such cultures is time-consuming and expensive, and there is the risk that chromosomal rearrangements may occur during extended fibroblast culture. Here we describe two procedures for the isolation from short-term lymphocyte cultures of metaphase chromosomes that are morphologically intact, suitable for flow cytometric analysis and purification, and have DNA of high molecular weight.