scholarly journals Generating Cloned Goats by Somatic Cell Nuclear Transfer—Molecular Determinants and Application to Transgenics and Biomedicine

2021 ◽  
Vol 22 (14) ◽  
pp. 7490
Author(s):  
Maria Skrzyszowska ◽  
Marcin Samiec
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Efficient Solutions ◽  
Genetically Engineered ◽  
Livestock Breeding ◽  
Future Challenges ◽  
Capra Aegagrus ◽  
The One

The domestic goat (Capra aegagrus hircus), a mammalian species with high genetic merit for production of milk and meat, can be a tremendously valuable tool for transgenic research. This research is focused on the production and multiplication of genetically engineered or genome-edited cloned specimens by applying somatic cell nuclear transfer (SCNT), which is a dynamically developing assisted reproductive technology (ART). The efficiency of generating the SCNT-derived embryos, conceptuses, and progeny in goats was found to be determined by a variety of factors controlling the biological, molecular, and epigenetic events. On the one hand, the pivotal objective of our paper was to demonstrate the progress and the state-of-the-art achievements related to the innovative and highly efficient solutions used for the creation of transgenic cloned does and bucks. On the other hand, this review seeks to highlight not only current goals and obstacles but also future challenges to be faced by the approaches applied to propagate genetically modified SCNT-derived goats for the purposes of pharmacology, biomedicine, nutritional biotechnology, the agri-food industry, and modern livestock breeding.

48 EFFECTS OF TRICHOSTATIN A ON DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 142 ◽  
Author(s):  
D. Iwamoto ◽  
K. Saeki ◽  
S. Kishigami ◽  
A. Kasamatsu ◽  
A. Tatemizo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Mammalian Species ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Embryonic Genome ◽  
Blastocyst Rate

Although cloning by somatic cell nuclear transfer (SCNT) has been achieved in various mammalian species, its efficiency has been very low (Han et al. 2003 Theriogenology 59, 33–44). Successful cloning requires conversion from differentiated donor nuclei to embryonic nuclei after transfer of the somatic nuclei into enucleated oocytes. Reprogramming of the transferred somatic nuclei must be completed by the time when normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, both full-term development and pre-implantation development of mouse SCNT embryos were significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The objective of this study was to investigate the effects of TSA on the development of bovine SCNT embryos. Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. The cells were electro-fused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF medium until 168 h post-activation (hpa). The NT embryos were exposed to 0 (control), 5, 50, and 500 nM TSA from the start of activation to 48 hpa. Experiments were repeated 3 times, and the data were analyzed with Fisher's PLSD test following ANOVA. The cleavage rates were the same among the groups (60 to 80%; P >0.05). However, the blastocyst rate of NT embryos treated with 50 nM TSA was higher than that of control embryos (40% vs. 19%, respectively; P < 0.05). On the other hand, the blastocyst rate was lower with 500 nM TSA than with 5 or 50 nM TSA (7% vs. 33% or 40%; P < 0.05). These data suggest that proper TSA treatment after somatic cloning improves the rate of development of bovine cloned embryos to the blastocyst stage. Further research is needed to examine whether NT embryos derived from different cell lines or types have similar susceptibility to TSA.

Somatic cell nuclear transfer in pigs: recent achievements and future possibilities

2007 ◽  
Vol 19 (2) ◽  
pp. 403 ◽  
Author(s):  
Gábor Vajta ◽  
Yunhai Zhang ◽  
Zoltán Macháty
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Direct Evidence ◽  
Research Area ◽  
Genetically Engineered ◽  
Transgenic Pigs ◽  
Actual Performance ◽  
Donor Cells

During the past 6 years, considerable advancement has been achieved in experimental embryology of pigs. This process was mainly generated by the rapidly increasing need for transgenic pigs for biomedical research purposes, both for future xenotransplantation to replace damaged human organs or tissues, and for creating authentic animal models for human diseases to study aetiology, pathogenesis and possible therapy. Theoretically, among various possibilities, an established somatic cell nuclear transfer system with genetically engineered donor cells seems to be an efficient and reliable approach to achieve this goal. However, as the result of unfortunate coincidence of known and unknown factors, porcine embryology had been a handicapped branch of reproductive research in domestic animals and a very intensive and focused research was required to eliminate or minimise this handicap. This review summarises recent achievements both in the background technologies (maturation, activation, embryo culture) and the actual performance of the nuclear replacement. Recent simplified methods for in vivo development after embryo transfer are also discussed. Finally, several fields of potential application for human medical purposes are discussed. The authors conclude that although in this early phase of research no direct evidence can be provided about the practical use of transgenic pigs produced by somatic cell nuclear transfer as organ donors or disease models, the future chances even in medium term are good, and at least proportional with the efforts and sums that are invested into this research area worldwide.

Resiliency of equid H19 imprint to somatic cell reprogramming by oocyte nuclear transfer and genetically induced pluripotency†

2019 ◽  
Author(s):  
Mikhael Poirier ◽  
Olivia Eilers Smith ◽  
Jacinthe Therrien ◽  
Nathia Nathaly Rigoglio ◽  
Maria Angélica Miglino ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Ctcf Binding ◽  
Differentially Methylated Region ◽  
Cell Reprogramming ◽  
Biallelic Expression ◽  
H19 Gene ◽  
Induced Pluripotent

Abstract Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetically stable after reprogramming in this species. In light of this, we aimed at characterizing the equid H19 gene to determine whether imprinting is altered in somatic cell nuclear transfer (SCNT)-derived conceptuses and induced pluripotent stem cell (iPSC) lines using the mule hybrid model. A CpG-rich region containing five CTCF binding sites was identified upstream of the equine H19 gene and analyzed by bisulfite sequencing. Coupled with parent-specific and global H19 transcript analysis, we found that the imprinted H19 remains monoallelic and that on average the methylation levels of both parental differentially methylated regions in embryonic and extra-embryonic SCNT tissues and iPSC lines remained unaltered after reprogramming. Together, these results show that, compared to other species, equid somatic cells are more resilient to epigenetic alterations to the H19-imprinted locus during SCNT and iPSC reprogramming.

43 PATTERN OF NUCLEOLI FORMATION IN RACCOON-PORCINE INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 169
Author(s):  
Y. H. Nam ◽  
Y. Jeon ◽  
S. A. Cheong ◽  
S. S. Kwak ◽  
S. H. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Housekeeping Genes ◽  
Blastocyst Stage ◽  
Control Group ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Embryonic Genome

Recently, great focus has been on the rescue of endangered animals through somatic cell nuclear transfer (SCNT). Because it is difficult to obtain the oocytes of endangered species, interspecies SCNT (iSCNT) methods have been attempted. Numerous iSCNT embryos have shown unsuccessful development due to aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). In particular, aberrant EGA may cause the arrest of nucleoli formation and developmental block in embryos. According to this concept, we performed raccoon iSCNT using porcine oocytes and analyzed iSCNT embryo development pattern and formation of nucleoli. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. Cleavage and blastocyst formation were evaluated under a stereomicroscope at 48 and 168 h post-activation (hpa), respectively. To confirm the formation of nucleoli, which can be detected by C23 antibody labeling in many mammalian species, C23 immunocytochemistry was performed at 48 and 72 hpa. A total of 158 iSCNT embryos were cultured; 68.5% of the raccoon iSCNT embryos were cleaved at 48 hpa (1-cell stage: 9.7%; 2-cell stage: 14.4%; 4-cell stage: 34.1%; 6-cell stage: 12.7%; 8-cell stage: 7.3%; fragmented: 21.8%). But, the embryos seen as 5- to 8-cell stage did not have the same number of nuclei as their blastomere number. When raccoon iSCNT embryos were stained by Hoechst 33342, 5- to 8-blastomere raccoon iSCNT embryos had only 4 nuclei. The raccoon iSCNT embryos did not develop past the 4-cell stage and failed to form blastocysts. In the control group, 65.2% of pig SCNT embryos were cleaved at 48 hpa (1-cell stage: 8.0%; 2-cell stage: 4.2%; 4-cell stage: 23.6%; 6-cell stage: 13.6%; 8-cell stage: 23.8%; fragmented: 26.8%), and 10.0% of pig SCNT embryos developed to blastocysts. In raccoon iSCNT embryos, raccoon nuclei failed to form nucleoli at 48 and 72 hpa. By contrast, pig SCNT embryos showed 18.8 and 87.9% nucleoli formation at 48 and 72 hpa. Our results demonstrate that 4-cell-stage embryos of raccoon-porcine hybrid embryos may be produced by SCNT methods. The pig oocytes partly supported the remodeling and reprogramming of the raccoon somatic cell nuclei, but they were unable to support nucleoli formation. Moreover, aberrant nucleoli formation caused the unsuccessful development of raccoon SCNT embryos to the blastocyst stage. This work was supported by a grant from the Next Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

scholarly journals Establishment of bovine expanded potential stem cells

2021 ◽  
Vol 118 (15) ◽  
pp. e2018505118
Author(s):  
Lixia Zhao ◽  
Xuefei Gao ◽  
Yuxuan Zheng ◽  
Zixin Wang ◽  
Gaoping Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Adult Individual ◽  
Developmental Potential ◽  
Cell Lineages

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

Somatic cell nuclear transfer

2004 ◽  
Vol 44 (11) ◽  
pp. 1101
Author(s):  
J. R. Hill
Keyword(s):  
Somatic Cell ◽  
Gene Function ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Front Page ◽  
Nuclear Reprogramming ◽  
Gene Insertion ◽  
Early Results ◽  
Quantum Leap

Nuclear transfer research became front-page news when the birth of Dolly, the cloned ewe, was reported by Ian Wilmut and Keith Campbell in 1997. Since Dolly’s birth, offspring from many other species have been produced using somatic cell nuclear transfer. While Dolly’s birth transformed embryology research, her death in February 2003 marked the beginning of the next phase of research and development. This period will determine the scale of the commercial and societal benefits that accrue from somatic cell nuclear transfer and transgenics. Proof of concept for many of the potential benefits of somatic cell nuclear transfer has already been demonstrated. Desirable genotypes have been cloned, further insights into the nuclear reprogramming process have been achieved, and precision gene insertions/deletion has been demonstrated. It is likely that nuclear transfer can be adapted to ‘copy’ individuals from any mammalian species. Offspring have been produced using cells from sheep, mice, cattle, goats, pigs, rabbits and a cat. It appears very likely that copying of other species such as horses will follow shortly. However, early results from monkeys suggest that somatic cell nuclear transfer in primates may require further intensive study before the likelihood of success can be predicted. The nuclear transfer process is far less efficient at producing healthy offspring than the natural process of combining a sperm with an egg. Fewer normal embryos, fetuses and offspring are produced from somatic cell nuclear transfer than from other assisted breeding techniques. The reasons for this appear to be related to abnormal expression of key developmental genes. Many of these genes are imprinted genes, which rely on correct methylation patterns of the genome that are established in the first week of life. Research into this area not only aids further development of the nuclear transfer technique but is also important for basic research into understanding the nuclear reprogramming process in mammals. The combination of nuclear transfer with gene insertion/deletion techniques has permitted a quantum leap in the efficiency of producing livestock with an additional ‘value adding’ gene. This has resulted in more economical production of animals that carry a specific valuable gene, such as a gene to enable production of novel or valuable proteins in their milk. Precision gene insertions or deletions will become more available in the near term so that this technique will become as important for testing gene function for agricultural applications as it is in mice for biomedical uses. Our challenge for the next decade is to fine-tune the somatic cell nuclear transfer technique so as to achieve more normal development rates. At the same time we need to increase the efficiency of targeted gene insertion or deletion so that the 2 techniques can be effectively combined to utilise the information on gene function created by livestock gene discovery programs.

scholarly journals Generation of Genetically Engineered Livestock Using Somatic Cell Nuclear Transfer

Reproduction ◽  
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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