Dataset Information

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

ABSTRACT: Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos.

SUBMITTER: Ruan D

PROVIDER: S-EPMC5830944 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

Ruan Degong D Peng Jiangyun J Wang Xiaoshan X Ouyang Zhen Z Zou Qingjian Q Yang Yi Y Chen Fangbing F Ge Weikai W Wu Han H Liu Zhaoming Z Zhao Yu Y Zhao Bentian B Zhang Quanjun Q Lai Chengdan C Fan Nana N Zhou Zhiwei Z Liu Qishuai Q Li Nan N Jin Qin Q Shi Hui H Xie Jingke J Song Hong H Yang Xiaoyu X Chen Jiekai J Wang Kepin K Li Xiaoping X Lai Liangxue L

Stem cell reports 20180111 2

Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullificatio ...[more]

PMID: 29337117

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Project description:BackgroundMelanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology.MethodFour CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis.ResultsFour vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R-/- and 10 MC3R+/- clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings.ConclusionA MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.

Dataset Information

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

Publications

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

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