Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts

Wan, Yongjie; Deng, Mingtian; Zhang, Guomin; Ren, Caifang; Zhang, Hao; Wang, Lizhong; Wang, Feng

doi:10.1002/cbin.10540

Cited by 15 publications

(5 citation statements)

References 44 publications

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“…Generally, imprinting disorder results in the developmental abnormalities of cloned animals and low cloning efficiency [ 4 ]. Here, we demonstrated that the imprinting status in PFFs derived from the morphologically abnormal cloned fetuses was disrupted, coinciding with the previous report [ 20 ]. Notably, along with the smaller sizes of the abnormal cloned fetuses, the even lower methylation levels and more disrupted expression patterns of imprinting genes were observed.…”

Section: Discussionsupporting

confidence: 92%

Imprinting disorder in donor cells is detrimental to the development of cloned embryos in pigs

Song

Jiang

et al. 2017

Oncotarget

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Imprinting disorder during somatic cell nuclear transfer usually leads to the abnormality of cloned animals and low cloning efficiency. However, little is known about the role of donor cell imprinting in the development of cloned embryos. Here, we demonstrated that the imprinting (H19/Igf2) in porcine fetus fibroblasts derived from the morphologically abnormal cloned fetuses (the abnormal imprinting group) was more hypomethylated, and accordingly, significantly higher H19 transcription and lower Igf2 expression occurred in comparison with those in fibroblasts derived from morphologically normal cloned fetuses (the normal imprinting group) or donor fetus fibroblasts (the control group). When these fibroblasts were used as donor cells, the abnormal imprinting group displayed an even lower imprinting methylation level, in correspondence to the significantly downregulated expression of Dnmt1, Dnmt3a and Zfp57, and a markedly reduced blastocyst rate, while the normal imprinting group took on the similar patterns of imprinting, gene expression and embryo development to the control group. When 5-aza-dC was applied to reduce the fibroblasts imprinting methylation level in the normal imprinting group, cloned embryos displayed the more severely impaired imprinting and significantly lower blastocyst rate. While the upregulated H19 transcription in the abnormal imprinting group was knocked down, the imprinting statuses were partly rescued, and the cleavage and blastocyst rates significantly increased in cloned embryos. In all, donor cell imprinting disorder reduced the developmental efficiency of cloned embryos. This work provides a new insight into understanding the molecular mechanism of donor cells regulating the cloned embryo development.

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Section: Discussionsupporting

confidence: 92%

Imprinting disorder in donor cells is detrimental to the development of cloned embryos in pigs

Song

Jiang

et al. 2017

Oncotarget

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“…In the previous study, PDX1 ‐modified pigs have been generated by SCNT, which strategy is commonly used to generate mutant pigs (Kang et al, 2017). Although SCNT enables to generate mutant pigs carrying desired mutations, the use of this technique is associated with small litter sizes, low viability, and abnormal epigenetic regulation of resulting offsprings (Kurome et al, 2013; Wan et al, 2016; Zhao, Whyte, & Prather, 2010). In this study, we used electroporation mediated gene editing to establish viable founder generations rescued by mosaicism and carrying PDX1 mutations in their germline to provide monoallelic/biallelic F1 generations.…”

Section: Discussionmentioning

confidence: 99%

Generation of viable PDX1 gene‐edited founder pigs as providers of nonmosaics

Tanihara

Hirata

Nguyen

et al. 2020

Molecular Reproduction Devel

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Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy.One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus. K E Y W O R D SCRISPR/Cas9, diabetes mellitus, electroporation, IVF, PDX1-mutant pigs SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section.

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“…Quantitative Real-Time PCR (qRT-PCR) was implemented using a previous researched method [ 24 ]. The relative mRNA expression levels of target genes were calculated using the comparative 2 −ΔΔCt method after normalization to GAPDH control.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Transcriptome Analysis of mRNA Expression Patterns of Early Embryo Development in Goat under Hypoxic and Normoxic Conditions

Wan

Deng

et al. 2021

Biology

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It has been reported that hypoxic environments were more suitable for the in vitro development of mammalian embryos, but the underlying mechanisms were still unclear. In the present study, RNA-seq was performed to compare 8-cell-stage and blastocyst-stage goat embryos under hypoxic and normoxic conditions; zygotes were checked at 72 and 168 h to 8-cell stage (L8C) and blastocyst stage (LM) in hypoxic conditions and 8-cell stage (H8C) and blastocyst stage (HM) in normoxic conditions. In the H8C and L8C groups, 399 DEGs were identified, including 348 up- and 51 down-regulated DEGs. In the HM and LM groups, 1710 DEGs were identified, including 1516 up- and 194 down-regulated DEGs. The expression levels of zygotic genes, transcription factors, and maternal genes, such as WEE2, GDF9, HSP70.1, BTG4, and UBE2S showed significant changes. Functional enrichment analysis indicated that these DEGs were mainly related to biological processes and function regulation. In addition, combined with the pathway–gene interaction network and protein–protein interaction network, twenty-two of the hub genes were identified and they are mainly involved in energy metabolism, immune stress response, cell cycle, receptor binding, and signal transduction pathways. The present study provides comprehensive insights into the effects of oxidative stress on early embryo development in goats.

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Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts

Cited by 15 publications

References 44 publications

Imprinting disorder in donor cells is detrimental to the development of cloned embryos in pigs

Imprinting disorder in donor cells is detrimental to the development of cloned embryos in pigs

Generation of viable PDX1 gene‐edited founder pigs as providers of nonmosaics

Comprehensive Transcriptome Analysis of mRNA Expression Patterns of Early Embryo Development in Goat under Hypoxic and Normoxic Conditions

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