Eif1ad3 as a key regulator during zygotic genome activation

Eif1ad3 with its homologs, including Eif1ad4, Eif1ad6, Eif1ad7, etc., which are barely studied before, are found to be highly translated during ZGA and regulating the translational activity of critical genes, especially for ribosome assembly. Knocking down Eif1ad3 leads to defective ZGA and further preimplantation development. These data identified Eif1ad3 as an indispensable factor governing mouse embryo development at ZGA. Overall design: For Eif1ad3 and its variance knockdown experiments, 1-Cell embryo were collected from CF1 (envigo) microinjected with control (only RfxCas13d-IVT mRNA) or Eif1adKD (gRNA+RfxCas13d-IVT mRNA). Embryos from control and knockdown groups were further cultured until blastocyst to observe the development. For RNA-Seq, 2-cell stage (control and knockdown groups) were collected. To generate Eif1ad3-6xHis mRNA, total RNA from 2-Cell embryos was extracted from a pool of mouse embryos, followed by first strand cDNA synthesis. To overexpress Eif1ad3, in vitro transcribed mRNAs (IVT-mRNAs) were microinjected into zygotes at final concentration of 200 ng/uL. RIP experiments were conducted using the EZ-Magna RIP Kit (Millipore Corporation, Billerica, MA). Briefly, 2-Cell stage embryos (over expressed 200 cells and control 200 cell per replicate) were harvested, lysed and then incubated with RIP buffer containing magnetic beads conjugated with anti-His tag antibody (Millipore) or corresponding negative control IgG (Millipore). After the antibody was recovered using protein A/G beads, the purified RNA were used to perform RNA-seq.