Sequential X-chromosome reactivation and inactivation in cell hybrids between murine embryonal carcinoma cells and female rat thymocytes.

By means of a 5-bromodeoxyuridine (BrdU) incorporation and an acridine orange fluorescence staining method together with [3H]thymidine ([3H]TdR) autoradiography, we studied the chronology of X-chromosome replication in newly formed cell hybrids between the hypoxanthine phosphoribosyl transferase (HPRT)-deficient OTF9-63 murine embryonal carcinoma (EC) cell with 43 +/- chromosomes and the female rat thymocyte having 42 chromosomes. Most near-tetraploid hybrid cells retained all chromosomes from both parents including one mouse X (XM) and two rat X (XR) chromosomes throughout the period of this study. Data showing changes in the chronology of X-chromosome replication obtained here were indicative of reactivation of the inactive X chromosome from the rat thymocyte, and de novo X-inactivation of one or two chromosomes. The extinction of lymphocyte phenotypes from the hybrids and their subsequent differentiation to the cell type resembling endoderm found in the peri-implantation mouse embryo apparently occurred in parallel with the above changes. These hybrids also showed an interesting possibility of preferential reinactivation of the reactivated XR chromosome in the early stages after cell fusion.