Establishment of Functional Genomics Pipeline in Mouse Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9

The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. Regulation of epiblast gene expression is poorly understood due to the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of embryonic stem cells (ESCs), we generated and characterized epiblast-like tissue in three-dimensional (3D) culture. We identified significant genome-wide expression changes in this epiblast-like tissue. Additionally, we identified the significance of the Fgf/Erk and ectoderm formation pathways, using the bioinformatics resource IPA and DAVID. We first focused on Fgf5, which ranked in the top 10 among discovered genes. Toward functional analysis of Fgf5, we developed efficient methods of genome engineering (CRISPR/Cas9) and RNA interference (RNAi). Notably, we show one-step generation of an Fgf5 reporter line, null and in/del mutants. Furthermore, mutation types correlated well with CRISPR/Cas9 activity. For time- and dose-dependent depletion of Fgf5 over the course of development, we generated an ESC line harboring a drug-inducible short hairpin RNA cassette integrated by the Tol2 transposon system (pRNAi). Our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics. The duplicated samples for each of at day0 (ESC) and day 3 (epiblast-like tissue) during SFEBq (serum-free floating culture of embryoid body-like aggregates with quick reaggregation) treatment were analyzed.