Comparison of Gene Expression and Genome-Wide DNA Methylation Profiling between Phenotypically Normal Cloned Pigs and Conventionally Bred Controls

Fei Gao,Anders Lade Nielsen,Lin Lin,Lars Bolund,Yonglun Luo,Huanming Yang,Shengting Li,Charlotte Brandt Sørensen,Xiuqing Zhang,Yutao Du,Gábor Vajta,Jian Li,Jun Wang

doi:10.1371/journal.pone.0025901

Fei Gao, Anders Lade Nielsen + Show 11 more

Open Access

https://doi.org/10.1371/journal.pone.0025901

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Abstract

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.

Highlights

DNA methylation occurs at most CpG dinucleotides in the mammalian genome [1]
In order to identify the mechanisms related to the inefficiency of Somatic Cell Nuclear Transfer (SCNT) technology, we compared global gene expression profiles between cloned animals and conventionally bred controls
Few studies have been conducted on full-term cloned pigs that have survived the full pressure from SCNT and embryonic development

Summary

Introduction

DNA methylation occurs at most CpG dinucleotides in the mammalian genome [1]. Reprogramming of DNA methylation is essential for early embryonic development, as genome-wide reprogramming of DNA methylation ensures removal of zygotic methylation marks in the nucleus and reestablishment of a different set of marks important for generating toti- and pluripotency [2]. DNA methylation is one of the most studied epigenetic regulatory mechanisms which plays a key role in gene expression regulation It is essential for establishing genomic imprints for tissue-specific differentiation in the early stage embryo. SCNT has been successfully applied in many mammalian animals including pigs, and many efforts have been endeavored to simplify the procedure and to increase the efficiency. This is exemplified in an approach named ‘‘handmade cloning (HMC)’’ [6,7] that is completely free of micromanipulations as well as in an approach involving pretreatment of the oocytes with high hydrostatic pressure (HHP) to improve developmental competence [8]. To produce viable offspring by SCNT, a drastic spatial and temporal remodeling of gene expression, invovling DNA methylation, is required to mimic the embryonic development in vivo [2]

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