X-Linked EGFP Reporter as a Tool to Examine X-Chromosome Inactivation in Mouse Embryos and Embryonic Fibroblasts

This study aimed to establish a model for investigating X chromosome inactivation using transgenic mouse strains expressing green fluorescent protein (GFP). The D4/XGFP-Tg (XGFP) strain carries the GFP transgene on the X chromosome; therefore, due to random X chromosome inactivation, female offspring from crosses between XGFP males and CD-1 females exhibit mosaic GFP expression. In contrast, the B5/EGFP-Tg (EGFP) strain harbours autosomal integration of the same reporter construct, resulting in uniform GFP expression in progenies. Analysis of CD-1 × XGFP attached blastocysts revealed strong GFP expression in giant trophoblast cells and primordial germ cells (PGCs) at E6.5, demonstrating paternal X-chromosome reactivation. In 14.5-day-old CD-1 × XGFP female embryos and CD-1 × EGFP embryos, intense CAG promoter-driven GFP signals were detected in the brain, heart, gonads, somites, and limbs. In line with random X-chromosome inactivation, only 56% of embryonic fibroblast cells, derived from CD-1 × XGFP female embryos, exhibited GFP expression. These findings validate that CD-1 × XGFP mice represent a valuable in vivo model for studying X chromosome inactivation during early embryonic development and PGC specification. Furthermore, CD-1 × XGFP embryonic fibroblasts represent a valuable in vitro model for investigating the molecular mechanisms governing X-chromosome activation and inactivation.