[Analysis of expression of parental alleles Xist and Gla in interspecific embryonic hybrid cells during induced in vitro inactivation of X-chromosomes].

The results of in situ hybridization with labeled species specific and X-chromosome-specific probes suggest that hybrid cells obtained by fusion of Mus musculus embryonic stem cells (genotype XY) and splenocytes of M. caroli females contain two parental X-chromosomes. In five clones of hybrid cells, differentiation was induced in embryoid bodies in vitro, which was accompanied by inactivation of one of X-chromosomes. We analyzed the expression of Xist and Gla alleles in the embryoid bodies using RT-PCR with an account that expression of locus Xist is one of key events in X-chromosome inactivation, while gene Gla was used as a marker of active X-chromosome. Identification of allele transcripts of loci Xist and Gla was based on restriction polymorphism between M. musculus and M. caroli that we had described. Transcripts of both parental alleles of loci Xist and Gla were present in the embryoid bodies of all studied hybrid clones. No preferential inactivation of M. musculus or M. caroli X-chromosome was found in the tested embryonic hybrid cells despite the initial differences in ontogenetic status between X-chromosomes of embryonic stem cells and splenocytes.

[Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells].

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.