Journal
PROTEIN & CELL
Volume 13, Issue 2, Pages 102-119Publisher
OXFORD UNIV PRESS
DOI: 10.1007/s13238-021-00890-3
Keywords
paternal imprints; androgenetic haploid ESCs; DMRs; semi-cloned mice; alternative 2i
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Funding
- Genome Tagging Project
- Chinese Academy of Sciences
- National Key Research and Development Program of China
- National Natural Science Foundation of China [2019YFA0109900, 2020YFA0509000, XDB19010204, QYZDJ-SSW-SMC023, 31821004, 32030029, 31730062]
- Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation
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The use of TSa2i protocol results in the establishment of AG-haESCs that can long-term maintain paternal imprints and show improved developmental potential compared to early-passage cells.
The use of two inhibitors of Mek1/2 and Gsk3 beta (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that the use of a two-step a2i (alternative two inhibitors of Src and Gsk3 beta, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2i-treated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can long-term maintain paternal imprints.
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