Trask B J, van den Engh G, Christensen M, Massa H F, Gray J W, Van Dilla M
Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, California 94550.
Somat Cell Mol Genet. 1991 Mar;17(2):117-36. doi: 10.1007/BF01232970.
We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequence as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.
我们报告了使用流式核型分析和荧光原位杂交（FISH）来鉴定体细胞杂种中的人类染色体。人类染色体的身份、DNA含量和相对频率源自流式核型，即通过双光束流式细胞术测量染色体的Hoechst和嗜色菌素荧光强度。通过比较杂交细胞系及其人类供体细胞的流式核型中人类染色体的峰位置来评估染色体完整性。当无法获得人类供体细胞时，将杂交系中人类染色体的峰位置与正常个体中的峰位置范围进行比较。使用流式核型中峰的体积监测培养中生长的亚克隆或杂种中人类染色体的相对频率。以生物素化的人类基因组DNA或染色体特异性重复序列作为探针的FISH与流式核型分析结合使用，以确认杂种中人类染色体的数量。一些小的重排可通过流式核型分析检测到，而FISH检测不到。另一方面，人类和啮齿动物染色体之间的易位可通过FISH检测到，而流式核型分析并不总是能检测到。流式核型分析和FISH被用于鉴定其他实验室捐赠的100多个杂种细胞系。展示了一组可用于构建富含染色体的基因文库的杂种细胞系。在这一组中，通过这些技术判断，24种人类染色体类型中的每一种都存在且完整，存在于几乎不包含或不包含其他人类物质的细胞系中。
