Global DNA methylation profiles of buffalo (Bubalus bubalis) preimplantation embryos produced by handmade cloning and in vitro fertilization

Abstract

Somatic cell nuclear transfer technique (SCNT) has proved to be an outstanding method of multiplication of elite animals but accompanied with low efficiency and live birth rate of cloned animals. Epigenetic alterations of DNA has been one of the culprits behind this issue. Cloned embryos are found to deviate slightly from regular pattern of demethylation and re-methylation at the time of nuclear reprogramming and embryonic development when compared with embryos produced by in vitro fertilization (IVF). Thus, the present study was aimed at evaluating global DNA methylation profiles of cloned embryos at 2-cell, 8-cell and blastocyst stages and compare it with corresponding stages of embryos produced by IVF by using MeDIP-Sequencing on Illumina-based platform. We found out that cloned embryos exhibited significantly different DNA methylation pattern as compared to IVF embryos with respect to distribution of differentially methylated regions in different components of genome, CpG islands distribution and methylation status, gene ontological profiles and pathways affected throughout the developmental stages. The data generated from MeDIP-Seq was validated at blastocyst stage cloned and IVF embryos by bisulfite-sequencing PCR on five randomly selected gene regions.

Epigenetic manipulation to improve mouse SCNT embryonic development

“…In 2018, Gao et al found there were re-methylated regions enriched in LTR elements in mouse cloned embryos during the 2-cell and 4-cell stages, and these re-methylated regions resulted in improper expression of genes and retrotransposons that are important for ZGA ( Gao et al, 2018b ). In addition to mice, other mammals also have abnormal DNA methylation patterns during pre-implantation as revealed by 5mC antibody-based immunostaining, bisulfite sequencing, WGBS, and MeDIP-sequencing ( Dean et al, 2001 ; Beaujean et al, 2004 ; Li et al, 2014 ; Zhang et al, 2016 ; Song et al, 2017 ; Wang et al, 2020b ; Deng et al, 2020 ; Malpotra et al, 2022 ). From the aforementioned SCNT studies, overexpression of Kdm4d and Xist KO may also help SCNT embryos through the DNA methylation barrier.…”