Bivariate flow karyotyping, sorting, and peak assignment of all rat chromosomes

A bivariate flow cytometric rat karyotype was established from second- and third-passage Copenhagen (Cop) rat embryo cell cultures. Chromosome suspensions from such cells (2n = 42 chromosomes) yielded bivariate flow karyotypes composed of 14-18 peaks, 10 of which were sortable into pools of single chromosome types. Conditions affecting resolution of peaks (including the length of colcemid treatment of cells and various combinations of fluorescent and nonfluorescent dyes) were optimized. Using chromosome suspensions from second-passage cultures of adult Cop male and female ear fibroblasts, peaks representing the X and Y chromosomes were identified. Assignment of chromosomes was accomplished by polymerase chain reactions of flow-sorted chromosomes using primers from mapped genes. Availability of this characterized rat flow karyotype should prove useful for assignment of genes to chromosomes as well as generation of chromosome-specific libraries in cloning assigned genes.