Studies on mural trophectoderm differentiation in mice.

To understand the gene expression dynamics of the mural trophectoderm (mTE) during implantation in mice, the mTE was isolated from B6D2F1 (DBA2JÃ?C57BL/6N) E3.5, E4.0, E4.5 and in vitro-cultured E4.5 blastocysts using a Bio-Cut Blade attached to a micromanipulator, and subjected to RNA-seq analysis. E3.5, E4.0 and E4.5 blastocysts were collected by flushing uteri and in vitro-cultured E4.5 blastocysts were produced by culturing the E3.5 blastocysts for 24 h in KSOM. The mTEs collected from 16 blastocysts were pooled as a sample, and three samples were prepared for each stage. In addition, to understand the significance of Cdx2 downregulation in the mTE during implantation, trophectoderm-specific Egfp or Cdx2 overexpression was performed by means of lentiviral vector-mediated gene transduction. B6D2F2 (BDF1Ã?BDF1) Egfp- and Cdx2-overexpressed blastocysts that had been transferred into pseudo-pregnant mice at 2.5 days post-coitum (dpc) were recovered by flushing uteri at 4.5 dpc. Six Egfp-overexpressed blastocysts and eight Cdx2-overexpressed blastocysts were individually subjected to RNA-seq analysis. Total RNA was extracted using an RNeasy Plus Micro Kit (QIAGEN) and cDNA libraries were generated using a SMART-seq HT PLUS Kit (Clontech). The indexed libraries were sequenced using an Illumina NextSeq 500 under 75-bp single-end conditions.