Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages

Abstract

Mouse embryonic stem (ES) cells are widely used in early development studies and for transgenic animal production; however, a stable karyotype is a prerequisite for their use. We derived 32 ES cell lines of outbred mice (129 9 BALB (1B), C57BL 9 1B, and DD 9 1B F1 hybrids). Pluripotency was assessed by utilizing stem-cell-marker gene expression, teratoma formation assays and the formation of chimeras. It was shown that only 21 of the 32 ES cell lines had a diploid modal number of chromosomes of 40. In these lines, the percentage of diploid cells varied from 30.3 to 78.9 %, and trisomy of chromosomes 1, 8 and 11 was observed in some cells in 16.7, 36.7 and 20.0 % of the diploid ES cell lines, respectively. Some cells had trisomy of chromosomes 6, 9, 12, 14, 18 and 19. In situ hybridization with an X chromosome paint probe revealed that 7 of the 11 XXcell lines had X chromosome rearrangements in some cells. Analysis of the methylation status of the Dlk1-Dio3 locus showed that imprinting was altered in 4 of the 18 ES cell lines. Thus, mouse ES cell lines are prone to chromosome abnormalities even at early passages. Therefore, routine cytogenetic and imprinting analyses are necessary for ES cell characterization.

Ascorbic acid improves pluripotency of human parthenogenetic embryonic stem cells through modifying imprinted gene expression in the Dlk1-Dio3 region

“…Menzorov et al . found that mouse ESCs can contribute into teratoma and even in chimera although their Dlk1-Dio3 imprinting control region is hypomethylated [ 44 ]. This result is similar to our findings, which confirmed that poor methylation seems not to disturb ESCs pluripotency, but in our results, important paternally expressed genes in this imprinting region were up-regulated and positively related to ESCs differentiation ability, which indicated that methylation is not the only regulation mode for these genes in the Dlk1-Dio3 region.…”

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

“…Mice were provided by the Center for Genetic Resources of Laboratory Animals (RFMEFI61914X0005, RFMEFI62114X0010) at ICG SB RAS. Cell culture was performed as described previously (Menzorov et al 2016…”

“…Experiments were performed in the Center for Genetic Resources of Laboratory Animals (RFMEFI61914X0005, RFMEFI62114X0010) at ICG SB RAS. The protocol was described earlier (Menzorov, Pristyazhnyuk, Kizilova, Yunusova, Battulin, Zhelezova, Golubitsa and Serov, 2016).…”

“…The protocol was described earlier (Menzorov, Pristyazhnyuk, Kizilova, Yunusova, Battulin, Zhelezova, Golubitsa and Serov, 2016). The experiments were performed at the Common Use Center Vivarium for Conventional Animals.…”

The necessity of cell banks

“…Это делает необходимым постоянный мони-торинг кариотипа. Показано, например, что ЭСК мыши даже на ранних пассажах культивирования могут иметь нестабильный состав хромосом (Menzorov et al, 2016).…”