Strategies for improvement of cloning by somatic cell nuclear transfer

2019 ◽  
Vol 59 (7) ◽  
pp. 1218 ◽  
Author(s):  
Xiaoyan Qiu ◽  
Xiong Xiao ◽  
Graeme B. Martin ◽  
Nan Li ◽  
Wenhui Ling ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Animal Species ◽  
Transgenic Animals ◽  
Cloning Efficiency ◽  
Developmental Problems ◽  
Donor Nucleus ◽  
Low Efficiency ◽  
Genomic Reprogramming

Somatic cell nuclear transfer (SCNT) is a powerful tool that is being applied in a variety of fields as diverse as the cloning and production of transgenic animals, rescue of endangered species and regenerative medicine. However, cloning efficiency is still very low and SCNT embryos generally show poor developmental competency and many abnormalities. The low efficiency is probably due to incomplete reprogramming of the donor nucleus and most of the developmental problems are thought to be caused by epigenetic defects. Applications of SCNT will, therefore, depend on improvements in the efficiency of production of healthy clones. This review has summarised the progress and strategies that have been used to make improvements in various animal species, especially over the period 2010–2017, including strategies based on histone modification, embryo aggregation and mitochondrial function. There has been considerable investiagation into the mechanisms that underpin each strategy, helping us better understand the nature of genomic reprogramming and nucleus–cytoplasm interactions.

Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes

2013 ◽  
Vol 25 (8) ◽  
pp. 1142 ◽  
Author(s):  
Insung Hwang ◽  
Yeon Woo Jeong ◽  
Joung Joo Kim ◽  
Hyo Jeong Lee ◽  
Mina Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Canis Lupus ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fusion Rate ◽  
Domestic Dogs ◽  
Domestic Dog ◽  
Canis Lupus Familiaris ◽  
Cloning Efficiency ◽  
Donor Cells

Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature’s diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P < 0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones’ inheritance of maternal domestic dog mitochondrial DNA.

scholarly journals Lessons Learned from Somatic Cell Nuclear Transfer

2020 ◽  
Vol 21 (7) ◽  
pp. 2314 ◽  
Author(s):  
Chantel Gouveia ◽  
Carin Huyser ◽  
Dieter Egli ◽  
Michael S. Pepper
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Lessons Learned ◽  
Epigenetic Reprogramming ◽  
Area Of Interest ◽  
Legal Implications ◽  
Low Efficiency

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.

scholarly journals Somatic nuclear transfer in livestock species

2001 ◽  
Vol 44 (4) ◽  
pp. 351-364 ◽  
Author(s):  
B. Kühholzer ◽  
G. Brem
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Transgenic Animals ◽  
Cell Types ◽  
Powerful Technique ◽  
Livestock Species ◽  
Recent Advances ◽  
Somatic Nuclear Transfer ◽  
Reported Data

Abstract. In this review, we discuss the recent advances in somatic cell nuclear transfer (NT) in sheep, cattle, goats, swine and rabbits. Numerous advances have been reported as this technique has developed over the last five years. In the first part of this review, we describe the reported data pertaining to each of the species mentioned above. Theories are offered to explain the different results seen between different species and cell types. One of the main aspects of somatic cell NT, the production of transgenic animals will also be reviewed. Future applications of this powerful technique are discussed. This review concludes with a discussion of some of the problems observed in animals produced using NT as well as possible Solutions for these challenges.

scholarly journals Somatic cell nuclear transfer: failures, successes and the challenges ahead

2019 ◽  
Vol 63 (3-4-5) ◽  
pp. 123-130 ◽  
Author(s):  
Marta Czernik ◽  
Debora A. Anzalone ◽  
Luca Palazzese ◽  
Mami Oikawa ◽  
Pasqualino Loi
Keyword(s):  
Endangered Species ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Transgenic Animals ◽  
Factors Affecting ◽  
Potential Applications ◽  
Mitochondrial Defects ◽  
Species Production

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before.

28 TREATMENT WITH SUBEROYLANILIDE HYDOXAMIC ACID OR SODIUM BUTYRATE ON PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DERIVED FROM KIDNEY CELLS OF HUMAN HEME OXYGENASE-1 TRANSGENIC PIG

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
K.-Y. Song ◽  
J.-H. Moon ◽  
E.-J. Park ◽  
S.-J. Kim ◽  
Y.-B. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Sodium Butyrate ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Heme Oxygenase 1 ◽  
Kidney Cells ◽  
Cloning Efficiency ◽  
Blastocyst Formation

Because somatic cell nuclear transfer (SCNT) is influenced by many factors concerning a series of various steps, the cloning efficiency is low in so many species and it seems to be more serious in production of transgenic (TG) animals. Reprogramming of donor nucleus is one of the important factors that affects the developmental competence of SCNT embryos, and several epigenetic remodelling drugs have been used to improve the cloning efficiency. In this study, we examined the effect of suberoylanilide hydoxamic acid (SAHA) or sodium butyrate (NaBu) treatment on the development of porcine SCNT embryos derived from kidney cells of TG pig. Fully confluent porcine kidney cells expressing the human heme oxigenase-1 gene were used for nuclear donor. For SCNT, matured oocytes with 1st polar body were enucleated, electrically fused, and activated 1 h after fusion (Song et al. 2009 Mol. Reprod. Dev. 76, 611–619). Then, SCNT embryos were incubated in postactivation medium [PA; porcine zygote medium-5 (PZM-5) supplemented with 0.5% dimethyl sulfoxide] for 4 h (control), PA with 0.4 μg mL–1 demecolcine for 4 h (Dc), PA with 0.5 μM SAHA for 9 h (SAHA), or PA with 1 mM NaBu for 9 h (NaBu). After postactivation treatment, SCNT embryos were cultured in fresh PZM-5 for 7 days. The embryos were examined for cleavage and blastocyst formation on Days 2 and 7, respectively (the day of SCNT was designated Day 0). Total cell number of blastocysts was examined by counting the number of nuclei stained with Hoechst 33342 under ultraviolet light. Complementary DNA synthesised with total RNA extracted from blastocysts were used for qRT-PCR to determine HDAC2, HDAC6, and GAPDH gene expression. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism version 5.01 (Graphpad Software, San Diego, CA, USA). The cleavage rates (77.0–82.9%) of treated embryos were not different from that of control embryos (79.0%). Blastocyst formation was slightly increased in Dc- (36/132, 27.3%), SAHA- (34/125, 28.6%), and NaBu- (36/133, 27.3%) treated embryos than in control embryos (32/128; 25.0%), but the difference was not significant. Total cell numbers (45.2–47.5) of treated embryos were not different from that of control embryos (51.8). Expression of HDAC2 was higher in SAHA-treated embryos than in control and Dc-treated embryos (P < 0.05), but it was not different from that of NaBu-treated embryos. The relative expression of HDAC6 transcript was increased in SAHA- and NaBu-treated embryos, but there was no significant difference among all groups. Although SAHA or NaBu did not improve the pre-implantational development of porcine SCNT embryos derived from kidney cells of TG pig as assessed in this study, additional studies are needed to determine the effect of SAHA or NaBu on gene expression of pig TG embryos and developmental competency following embryo transfer according to the origin of donor cells. This study was supported by IPET (#311011-05-2-SB010), MOTIE (#10033839-2012-21) and the TS Corporation.

The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda–rabbit interspecies nuclear transfer

Zygote ◽  
2004 ◽  
Vol 12 (4) ◽  
pp. 315-320 ◽  
Author(s):  
Man-Xi Jiang ◽  
Cai-Xia Yang ◽  
Li-Sheng Zhang ◽  
Yue-Liang Zheng ◽  
Shu-Zhen Liu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Giant Panda ◽  
Whole Cell ◽  
Intracytoplasmic Injection ◽  
Low Efficiency ◽  
Interspecies Cloning ◽  
Interspecies Nuclear Transfer

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda–rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda–rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p>0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda–rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer

2013 ◽  
Vol 80 (6) ◽  
pp. 630-635 ◽  
Author(s):  
Jun-Xue Jin ◽  
Suo Li ◽  
Qing-Shan Gao ◽  
Yu Hong ◽  
Long Jin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cloning Efficiency

Transgenesis in farm animals-A review

2017 ◽  
Vol 38 (02) ◽  
Author(s):  
Parul Mehta ◽  
Ankur Sharma ◽  
Ramakant Kaushik
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Transgenic Animals ◽  
Farm Animals ◽  
Zinc Finger Nucleases ◽  
The Novel ◽  
Transcription Activator ◽  
Human Welfare ◽  
Transgenic Livestock

For a number of decades, attempts have been made to successfully produce transgenic animals which have numerous applications in the biotechnology industry with the foremost emphasis on production of monoclonal antibodies and recombinant proteins of human welfare. Different techniques are adopted in order to produce transgenic farm animals which could be further used as bioreactors. The most common traditional transgenesis technique employed is Somatic Cell Nuclear Transfer (SCNT) using genetically modified somatic cells or stem cells as nuclear donors. This review article summarizes the merits and demerits of the techniques currently used to produce transgenic livestock with major emphasis on somatic cell nuclear transfer. In the end, a brief discussion is done about the novel methods adopted to produce transgenic animals like Zinc Finger Nucleases (ZFN), Transcription Activator-like Effector Nuclease (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR). It is expected that the new techniques developed would overcome the problems faced with existing traditional transgenesis methods.

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

2018 ◽  
Vol 45 (4) ◽  
pp. 1529-1540 ◽  
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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