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 sequences 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.
ASJC Scopus subject areas
- Cell Biology