82 Disruption of p53 in bovine somatic cells affected cloned embryonic development

Abstract

The p53 gene is a tumour suppressor gene with many roles in cellular reprogramming, senescence, or cancers. Knockdown of p53 in somatic cells improves generation of induced pluripotent stem cells and developmental competence of cloned embryos. In this study, we hypothesised that disruption of p53 in bovine somatic cells using CRISPR-Cas9 may improve reprogramming and result in increased blastocyst formation. For the study, ovary-derived immature bovine cumulus-oocyte complexes were matured in vitro. To prepare the donor cells with disruption of p53, primary cells from the skin tissue were transfected with spCas9 and single guide RNA for bovine p53. Two days later, genomic DNA was extracted and the mutation was evaluated on the target region using the T7E1 enzyme. Several single cell colonies were isolated to investigate the mutation and sequencing for mutation positive colonies was performed. One colony with high proliferative activity and heterozygotic mutation (−7 bp deletion) was selected and used as the donor cell for somatic cell nuclear transfer. The donor cell was microinjected into an enucleated oocyte. Reconstructed oocytes were fused by electrical pulse, activated by chemical stimulation, and cultured in chemically defined media for 7 days at 5% CO₂ and 5% O₂. Data were subjected to ANOVA and least significant different test to determine differences between experimental groups by using SAS software (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined when the P-value was less than 0.05. Blastocyst formation rate (13.5 ± 7.6%) from the mutated cell line was significantly lower than wild type cells (43.9 ± 12.1%). Moreover, total cell number (115.4 ± 13.3) of the blastocysts from knockout cells was significantly decreased compared with the control group (143.6 ± 22.5). Some randomly selected blastocysts derived from mutated cells were subjected to mutation assay to confirm deletion of p53. As expected, all the samples were positive to mutation. These data did not support our hypothesis that p53 disruption could improve blastocyst formation competence of bovine cloned embryos. In the future, the reason for impaired embryonic development of clones of p53 deleted somatic cells will be further investigated.

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.