69 Cloned bovine embryonic development derived from interferon tau knockout cells

Abstract

Interferon tau (IFNT), a type I interferon, is known as a key signal molecule during pregnancy in ruminants because of the necessity in maternal recognition of pregnancy. It is produced in trophectoderm cells of the elongation bovine conceptus at Day 13-21, and peak production is at Day 15 to 17 of pregnancy. In addition, other studies show that it can affect embryonic development and quality. In this study, the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. To proceed with this experiment, immature oocytes from ovaries at a local slaughterhouse were matured in vitro for 22h. To prepare the donor cell with IFNT knockout, somatic cells were transfected with Cas9 and single guide RNA targeting IFNT, and several single derived colonies with high proliferation were isolated for mutation assay. Finally, one colony that had mono-allelic mutation (4bp deletion) was selected and used as the donor cell for SCNT. A donor cell was injected into an enucleated oocyte. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation, and cultured for 7 days in chemically defined medium. For this study, control (n=94) and IFNT knockout groups (n=140) were compared with 4 replications. The results showed no significant difference between control and IFNT knockout groups not only in cleavage rate, but also in blastocyst formation rate (control: 15.7±8.3%, IFNT knockout group: 26.3±13.1%). In addition, the number of blastocyst cell was not different between control (88.2±27.0) and IFNT knockout group (88.0±21.1). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT, and all samples were positive for mutation. In conclusion, these data demonstrated that the disruption of IFNT did not affect embryonic development. In future study, we would transfer these embryos and check the effect of IFNT during pregnancy status. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.