Estudio de la función génica de PTGS2 durante el desarrollo embrionario bovino mediante la tecnología CRISPR/Cas9

ABSTRACT Prostaglandins (PGs) are important regulators of reproductive processes such as implantation or establishment of gestation. The limiting enzyme in PGs synthesis is prostaglandin-endoperoxide synthase 2 (PTGS2), whose expression increases considerably during pre-implantation embryonic development in ungulates. Prostaglandins produced by the conceptus have been suggested top lay autocrine roles required for embryo development. In order to study if conceptus-derived prostaglandins are required for its own development, we analysed the development of bovine embryos knock-out (KO) for PTGS2 using the CRISPR/Cas9 system. For this purpose, in vitro matured oocytes were divided into two groups, one microinjected with the mRNA coding for Cas9 (control group, Cas9, composed of WT embryos only), and the other microinjected with the guide RNA designed for PTGS2 as well (Cas9 + gRNA group composed partially of KO embryos). After in vitro fertilisation and culture until day 12, the rates of development to blastocyst (day 7) and survival to day 12 were analysed. In addition, cell lineages were identified by immunohistochemistry for hypoblast (SOX17), trophectoderm (CDX2) and epiblast (SOX2). Finally, embryos were genotyped by deep sequencing (Illumina). Developmental rates up to day 7 and survival until day 12 were similar between both groups. 6 of the 55 embryos microinjected with Cas9 + RNAgs were KO. These embryos showed no significant differences in size, complete hypoblast migration, epiblast survival and number of SOX2+ cells compared to WT and non-KO edited embryos. In conclusión, PTGS2 ablation does not affect embryonic development up to day 12 in bovine, suggesting that PGs produced by the embryo during this period are not necessary for its development up to such stage.