Lessons Learned from Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

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Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning

Reproduction ◽

10.1530/rep-07-0397 ◽

2008 ◽

Vol 135 (2) ◽

pp. 151-163 ◽

Cited By ~ 153

Author(s):

Heiner Niemann ◽

X Cindy Tian ◽

W Allan King ◽

Rita S F Lee

Keyword(s):

Somatic Cell ◽

Cell Nucleus ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Epigenetic Reprogramming ◽

Biological Research ◽

Foetal Development ◽

Research Activities ◽

Somatic Cell Nucleus

The birth of ‘Dolly’, the first mammal cloned from an adult donor cell, has sparked a flurry of research activities to improve cloning technology and to understand the underlying mechanism of epigenetic reprogramming of the transferred somatic cell nucleus. Especially in ruminants, somatic cell nuclear transfer (SCNT) is frequently associated with pathological changes in the foetal and placental phenotype and has significant consequences for development both before and after birth. The most critical factor is epigenetic reprogramming of the transferred somatic cell nucleus from its differentiated status into the totipotent state of the early embryo. This involves an erasure of the gene expression program of the respective donor cell and the establishment of the well-orchestrated sequence of expression of an estimated number of 10 000–12 000 genes regulating embryonic and foetal development. The following article reviews the present knowledge on the epigenetic reprogramming of the transferred somatic cell nucleus, with emphasis on DNA methylation, imprinting, X-chromosome inactivation and telomere length restoration in bovine development. Additionally, we briefly discuss other approaches towards epigenetic nuclear reprogramming, including the fusion of somatic and embryonic stem cells and the overexpression of genes crucial in the formation and maintenance of the pluripotent status. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realising the great potential of SCNT for basic biological research and for various agricultural and biomedical applications.

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Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts

Zygote ◽

10.1017/s0967199409990372 ◽

2010 ◽

Vol 18 (2) ◽

pp. 93-101 ◽

Cited By ~ 35

Author(s):

S. Kim ◽

J.H. Kim ◽

E. Lee ◽

Y.W. Jeong ◽

M.S. Hossein ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Embryonic Stem ◽

Donor Cell ◽

Mouse Embryonic Fibroblast ◽

Embryoid Bodies ◽

Transgenic Pigs ◽

Donor Cells ◽

Embryonic Antigen

SummaryThis study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1–60 and TRA-1–81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: β-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

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Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos

Cellular Physiology and Biochemistry ◽

10.1159/000487579 ◽

2018 ◽

Vol 45 (4) ◽

pp. 1529-1540 ◽

Cited By ~ 13

Author(s):

Zhiren Zhang ◽

Yanhui Zhai ◽

Xiaoling Ma ◽

Sheng Zhang ◽

Xinglan An ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Epigenetic Modification ◽

Expression Patterns ◽

Epigenetic Reprogramming ◽

Preimplantation Embryos ◽

Down Regulation ◽

Low Efficiency

Background/Aims: Aberrantly high levels of H3K4me3, caused by incomplete epigenetic reprogramming, likely cause a low efficiency of somatic cell nuclear transfer (SCNT). Smal molecule inhibitors aimed at epigenetic modification can be used to improve porcine SCNT embryo development. In this study, we examined the effects of MM-102, an H3K4 histone methyltransferase inhibitor, on porcine SCNT preimplantation embryos to investigate the mechanism by which H3K4 methylation regulated global epigenetic reprograming during SCNT. Methods: MM-102 was added to the SCNT embryos culture system and the global levels of various epigenetic modifications were measured by immunofluorescence (IF) staining and were quantified by Image J software. Relative genes expression levels were detected by quantitative real-time PCR. Results: MM-102 (75 μM) treatment reduced global H3K4, H3K9 methylation and 5mC levels especially at the zygotic gene activation (ZGA) and blastocyst stages. MM-102 treatment mainly down-regulated a series of DNA and histone methyltransferases, and up-regulated a number of hitone acetyltransferases and transcriptional activators. Furthermore, MM-102 treatment positively regulated the mRNA expression of genes related to pluripotency (OCT4, NANOG, CDX2) and apoptosis (BCL2). Conclusion: Down-regulation of H3K4me3 with MM-102 rescued aberrant gene expression patterns of a series of epigenetic chromatin modification enzymes, pluripotent and apoptotic genes at the ZGA and blastocyst stages, thereby greatly improving porcine SCNT efficiency and blastocyst quality, making them more similar to in vivo embryos (IVV).

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Advance in the Role of Epigenetic Reprogramming in Somatic Cell Nuclear Transfer-Mediated Embryonic Development

Stem Cells International ◽

10.1155/2021/6681337 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Xiaolei Zhang ◽

Shaorong Gao ◽

Xiaoyu Liu

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Embryonic Stem Cell Line ◽

Embryonic Stem ◽

Blastocyst Development ◽

Low Efficiency ◽

Nuclear Transfer Embryo

Somatic cell nuclear transfer (SCNT) enables terminally differentiated somatic cells to gain totipotency. Many species are successfully cloned up to date, including nonhuman primate. With this technology, not only the protection of endangered animals but also human therapeutics is going to be a reality. However, the low efficiency of the SCNT-mediated reprogramming and the defects of extraembryonic tissues as well as abnormalities of cloned individuals limit the application of reproductive cloning on animals. Also, due to the scarcity of human oocytes, low efficiency of blastocyst development and embryonic stem cell line derivation from nuclear transfer embryo (ntESCs), it is far away from the application of this technology on human therapeutics to date. In recent years, multiple epigenetic barriers are reported, which gives us clues to improve reprogramming efficiency. Here, we reviewed the reprogramming process and reprogramming defects of several important epigenetic marks and highlighted epigenetic barriers that may lead to the aberrant reprogramming. Finally, we give our insights into improving the efficiency and quality of SCNT-mediated reprogramming.

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Generation of Genetically Engineered Livestock Using Somatic Cell Nuclear Transfer

Reproduction ◽

10.1530/rep-21-0072 ◽

2021 ◽

Author(s):

Irina Polejaeva

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Genetic Manipulation ◽

Embryonic Stem ◽

Genetically Engineered ◽

Farm Animals ◽

Technological Advancement ◽

Genetic Mosaicism ◽

Low Efficiency

Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985 – 1996). The applications of technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of Somatic Cell Nuclear Transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996 – 2013), SCNT was the only method used for generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.

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Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd17543 ◽

2018 ◽

Vol 30 (10) ◽

pp. 1342 ◽

Cited By ~ 4

Author(s):

Zhao-Bo Luo ◽

Long Jin ◽

Qing Guo ◽

Jun-Xia Wang ◽

Xiao-Xu Xing ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Formation Rate ◽

Epigenetic Reprogramming ◽

Developmental Competence ◽

Abnormal Development ◽

Control Group ◽

Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

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A Novel Method for Somatic Cell Nuclear Transfer to Mouse Embryonic Stem Cells

Cloning and Stem Cells ◽

10.1089/clo.2005.7.265 ◽

2005 ◽

Vol 7 (4) ◽

pp. 265-271 ◽

Cited By ~ 27

Author(s):

Danièle Pralong ◽

Krzysztof Mrozik ◽

Filomena Occhiodoro ◽

Nishanthi Wijesundara ◽

Huseyin Sumer ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Novel Method

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Nuclear Donor Cell Lines Considerably Influence Cloning Efficiency and the Incidence of Large Offspring Syndrome in Bovine Somatic Cell Nuclear Transfer

Reproduction in Domestic Animals ◽

10.1111/rda.12140 ◽

2013 ◽

Vol 48 (4) ◽

pp. 660-664 ◽

Cited By ~ 10

Author(s):

J Liu ◽

Y Wang ◽

J Su ◽

Y Luo ◽

F Quan ◽

...

Keyword(s):

Somatic Cell ◽

Cell Lines ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Cloning Efficiency

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Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

Cellular Physiology and Biochemistry ◽

10.1159/000494598 ◽

2018 ◽

Vol 50 (4) ◽

pp. 1376-1397 ◽

Cited By ~ 5

Author(s):

Yanhui Zhai ◽

Zhiren Zhang ◽

Hao Yu ◽

Li Su ◽

Gang Yao ◽

...

Keyword(s):

Dna Methylation ◽

Somatic Cell ◽

Histone Modifications ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Histone H3 ◽

Dna Demethylation ◽

Epigenetic Reprogramming ◽

Expression Levels ◽

Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

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