QUANTITATIVE RT-PCR ASSAYS SHOW XIST RNA LEVELS ARE LOW IN MOUSE FEMALE-ADULT TISSUE, EMBRYOS AND EMBRYOID BODIES

We have investigated expression of the Xist gene in mouse female adult kidney, embryos and embryonic stem (ES) cells undergoing in vitro dif ferentiation as embryoid bodies. Using the quantitative RT-PCR single nucleotide primer extension (SNuPE) assay, we found that the amount of Xist RNA in adult kidney of three mouse strains was less than similar to 2000 transcripts per cell, with only modest differences between st rains carrying different Xce alleles, Female embryos 7.5 days post coi tum had the same number of Xist transcripts per cell as isogenic adult tissue, Using quantitative oligonucleotide hybridization assays after RT-PCR, we investigated Xist expression in ES lines heterozygous at t he Pgk-1 and Xist loci. We found that, while in most (XX) ES lines Xis t RNA levels increased during embryoid body formation, the levels seen were less than 10% those found in adult female kidney, In addition, w e found that the allelic ratio of Xist transcripts from reciprocal (XX ) ES cell lines differentiating in vitro was identical to that of isog enic 10.5 to 11.5 day female embryos. These latter results suggest tha t there is no pattern of preferential paternal imprinting during days 1 to 9 of in vitro differentiation of ES cells, However, the influence of the Xce locus on the randomness of X-inactivation in embyros seems to operate also in ES cell lines, Our overall conclusion is that the low levels of Xist RNA in female kidney, embryos and differentiating ( XX) ES cells are compatible only with models that do not require Xist RNA to cover the entire inactive X chromosome.