MR-121-D Sigma-AldrichEmbryoMax® KSOM Mouse Embryo Media

Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M(3)O). Transduction of M(3) O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.

PURPOSE: To assess the impact of embryonic stem cell culture medium (ESCM) on the pre- and post-implantation development of the mouse embryo, as a mammalian model, in comparison with the conventional culture medium, a potassium simplex optimized medium (KSOM). METHODS: Development in ESCM versus KSOM was compared in terms of embryo morphology, cleavage, cavitation, hatching, cell number, expression of TE and ICM transcription factors (Cdx2 and Oct4, respectively), implantation, and development in utero. RESULTS: An enriched medium like ESCM can be beneficial for in vitro embryo development when cultured from the 8-cell stage, as evidenced by promotion of blastocyst development with respect to cavity expansion, hatching, and cell division. Such benefits were not observed when embryos were cultured from the 2-cell stage. CONCLUSIONS: ESCM may augment in vitro embryo development from the 8-cell stage. Using different culture media at different stages may be beneficial to achieve more effective human in vitro fertilization.

Genetically modified mouse strains derived from embryonic stem (ES) cells have become essential tools for functional genomics and biomedical research. Large scale mutagenesis projects are producing libraries of mutant C57BL/6 (B6) ES cells to enable the functional annotation of every gene of the mouse genome. To realize the utility of these resources, efficient and accessible methods of generating mutant mice from these ES cells are necessary. Here, we describe a combination of ICR morula aggregation and a chemically-defined culture medium with widely available and accessible components for the high efficiency generation of germline transmitting chimeras from C57BL/6N ES cells. Together these methods will ease the access of the broader biomedical research community to the publicly available B6 ES cell resources.

l(2)dtl (lethal (2) denticleless), is an embryonic lethal homozygous mutation initially identified in Drosophila melanogaster that produces embryos that lack ventral denticle belts. In addition to nucleotide sequence, bioinformatic analysis has revealed a conservation of critical functional motifs among the human L2DTL, mouse L2dtl, and Drosophila l(2)dtl proteins. The function of the L2DTL protein in the development of mammalian embryos was studied using targeted disruption of the L2dtl gene in mice. The knock-out resulted in early embryonic lethality. L2dtl-/- embryos were deformed and terminated development at the 4-8-cell stage. Microinjection of a small interfering RNA (siRNA) vector (siRNA-L2dtl) into the two-cell stage nuclei of wild-type mouse embryos led to cell cycle progression failure, termination of cell division, and, eventually, embryonic death during the preimplantation stage. Morphological studies of the embryos 54 h after injection showed fragmentation of mitotic chromosomes and chromosomal lagging, hallmarks of mitotic catastrophe. The siRNA-L2dtl-treated embryos eventually lysed and failed to develop into blastocysts after 72 h of in vitro culturing. However, the embryos developed normally after they were microinjected into one nucleus of the two-celled embryos. The siRNA studies in HeLa cells showed that L2dtl protein depletion results in multinucleation and down-regulation of phosphatidylinositol 3-kinase, proliferating cell nuclear antigen, and PTTG1/securin, which might partially explain the mitotic catastrophe observed in L2dtl-depleted mouse embryos. Based on these findings, we conclude that L2dtl gene expression is essential for very early mouse embryonic development.