Derivation And Application Of Mouse Androgenetic Embryonic Stem Cells

Androgenetic embryos could not develop to term due to lacking of maternal genome. However,androgenetic embryonic stem (aES) cells could derive from androgenetic blastocysts in vitro. Thestability of epigenetic state in aES cells during culture resulte in their poor competency to generatechimeras. Here, we introduce “one step micromanipulation (OSM)” method to reconstructandrogenetic embryos efficiently and treat these embryos with CBHA. Compared to non-treatedandrogenetic embryos, treated ones possecessed higher efficiency of blastocysts in vitro. Furthermore, the CBHA teated aES (CaES) cells could generate healthy chimera by blastocyst-injection.By bisulfite genomic sequencing, we found that H19-DMR of CaES cells had altered. In addition,expression level of three imprinted genes (H19, Dio3and U2af1-rs1) in CaES cells normalized tothe standard of zygote-derived ESCs. CBHA brought in this molecular changes in aESs might bethe reason of improvement of their pluripotency, which could be a novel way to modifiy theepigenetic state of aES cells and helped us better understanding of epigenetic regulation.Androgenetic haploid embryos could be generated by injection of sperm head into enucleatedoocyte assisted with “OSM” method, which were employed to derive androgenetic haploidembryonic stem (ahES) cells. The ahES cells had similar colonies to zygotic-derived ESCs, andexpressed pluripotent markers. Besides, these cells could form teratoma and chimera, even hadgerm-line competency. DNA analysis of chimeras from ahES cells on embryonic day6.5(E6.5)indicated that haploid cells remained in this period. We established haploid epiSC-like cells bydifferentiation of ahES cells in vitro, which could differentiate to neural progenitor cells quickly.By FACS analysis, these neural progenitor cells could partially maintain haploidy for some time.We generate viable mice by intracytoplasmic injection of ahES (ICAI) cells. Then, we introducedNeorinto ahES cells by random mutation, and purify the target cells by G418selection and FACS.Eventually, we produce live transgenic mice by the ICAI procedure.