Obtaining embryonic stem cells with MEK1 and GSK3 inhibitors from parthenogenetically activated mouse embryos

Abstract

In mammalian full fetal development requires a genomic parental complementation. Due to exclusively maternal origin of parthenogenetic embryos, important paternal imprinted genes are not expressed and the whole development of conceptus is impaired. Obtaining a embryonic stem cells (ESC) line, from inner cell mass (ICM) of blastocyst, has the advantage of knowledge of the genotypic characteristics to the production or the reproduction of the future animal to be obtained. The parthenogenetic ESC (PGESC) have been used as a tool in animal biotechnology and basic studies of embryology, in order to understand events and mechanisms of the biology of development and cell niche concept. Recently, the use of inhibitors of cellular pathways for differentiation to maintain the pluripotent and undifferentiated status of ESC colonies is an alternative for less permissive strains of animals (mice) or to increase the effectiveness of the ESC derivation. Drugs responsible to inhibit MEK1 and GSK3 pathways are the most commonly used (ideally together) and named as "2i" ("two inhibitors"). Oocytes from transgenic mice (C57BL/6/EGFP strain) will be subjected to parthenogenetic activation protocol and cultured until the blastocyst stage. Zona pellucida will be removed from blastocysts and those will be cultured in 96-well plates containing medium for ESC derivation with inhibitors, on a monolayer of inactivated embryonic fibroblasts (MEF). Five to six days later the putative colonies will be evaluated for morphology and they will be transferred to another dish and cultured until the 9th day to a new passage. After 3rd passage (day 14 of culture) those lines with according growth will be characterized by morphological (nucleus: cytoplasm ratio and cell shape), cellular (karyotyping) and functional markers. The aims of this project are: to obtain ESC lines from in vivo fertilized (imprinting control) and parthenogenetically activated embryos (absence of paternal expression of imprinted genes alleles) to characterize epigenetic effects arising from derivation of the ESC itself. It will be assessed the expression of genes related to imprinting (Igf2, Igf2r and H19), to pluripotency (Oct4 and Sox2) and specific to blastocyst cell lines (Cdx2 and Oct4) in embryos and in stem cell lines derived from control (in vivo fertilization) and parthenogenetic embryos.