REACTIVATION OF AN INACTIVE HUMAN X-CHROMOSOME INTRODUCED INTO MOUSE EMBRYONAL CARCINOMA-CELLS BY MICROCELL FUSION WITH PERSISTENT EXPRESSION OF XIST

An inactive human X chromosome was introduced by microcell fusion into two mouse embryonal carcinoma cell lines, PSA1-TG8 and OTF9-63, each of which has a single X chromosome. The donor cell line was a mouse-hu man somatic cell hybrid, CF150, retaining one or more inactive human X chromosome(s) per cell as its only human element. Twenty hybrid clone s isolated retained EC morphology and contained the intact human Ii ch romosome(s) or its truncated derivative(s). Replication banding analys is showed that the introduced human Ii chromosome(s) or its derivative (s) replicated synchronously with other mouse chromosomes, suggesting reactivation of the human X chromosomal elements after transfer. Rever sal of inactivation was further confirmed by the expression of five hu man Ii-linked genes repressed in CF150, although the XIST (X inactive specific transcript) gene continued to be active, The level of XIST ex pression in our hybrid cells was almost identical to that of parental CF150 cells. Methylation status of 5' end of the active XIST gene vari ed considerably from almost full methylation to unmethylation in these hybrids. Thus, mouse EC cells used in this study were capable of alte ring methylation status of the human XIST gene in a manner lacking con sistency and unable to repress its transcription. Furthermore, we fail ed to obtain any positive evidence for the occurrence of X chromosome inactivation in differentiating monochromosome EC hybrids. Taken toget her, these findings suggest that the human X chromosome inactivation c enter including the XIST gene is unable to function effectively in mou se cells. (C) 1997 Academic Press.