Generation of female fibroblast cell lines in which Xist is conditionally and allele-specifically deleted from the inactive X chromosome.

Abstract

Dosage compensation is accomplished by X chromosome inactivation (XCI) in female cells of placental mammals. The silencing of either the paternal or the maternal X chromosome ensures the equal expression of X-linked genes in males and females. Xist is a critical gene involved in regulating XCI. The noncoding Xist RNA transcripts “coat” or “envelope” the entire X chromosome territory in cis, triggering the chromosome-wide gene silencing. The gene silencing status of the inactive X chromosome (Xi), once established, is stably maintained in somatic cells in an Xist-independent manner. Only small-scale gene reactivation was detected along the Xist-deficient Xi in somatic cells. Furthermore, gene reactivation after Xist removal is considered as a sporadic event, as different gene reactivation events were detected from different mutant cell lines generated in parallel. Such views remain to be further confirmed, as only a limited number of X-linked genes were analyzed in previous studies using the conventional allele-specific RT-PCR technique. In this project, I have generated five female fibroblast cell lines with conditional deletion of Xist allele-specifically on the Xi. The genotypes of the established cell lines were confirmed by PCR and RNA FISH analysis. The established cell lines can be used for future comprehensive profiling of gene reactivation pattern along the entire Xi.