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.

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

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.

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

2005 ◽  
Vol 7 (4) ◽  
pp. 265-271 ◽  
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

62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 139 ◽  
Author(s):  
S. Mitalipov ◽  
Q. Zhou ◽  
J. Byrne ◽  
W.-Z. Ji ◽  
D. Wolf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Lamin A ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Morula Stage

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

65 BLASTOCYSTS DERIVED FROM ADULT FIBROBLASTS OF RHESUS MONKEY (MACACA MULATTA) USING INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 191
Author(s):  
D. K. Kwon ◽  
J. T. Kang ◽  
S. J. Park ◽  
M. N. L. Gomez ◽  
S. J. Kim ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Somatic Cell ◽  
Macaca Mulatta ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Cell Stage ◽  
Nuclear Number

Interspecies somatic cell nuclear transfer (iSCNT) has alternatively chosen in primate SCNT because of the difficulty in collecting enough oocytes for research. The purpose of this experiment is to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissueofrhesus monkey (male, 6 years old) was collected by biopsy and fibroblasts were isolated. Immature COCs from cow ovaries were collected and matured in vitro in TCM-199. Squish enucleation was done in the presence of bisbenzimide and cytochalasin B. After enucleation, a single rhesus monkey somatic cell was injected into the perivitelline space of an enucleated oocyte through the slit in the zona pellucida made during enucleation. Subsequently, the rhesus monkey somatic cell and cow oocyte membranes were electrically fused. The nonactivated interspecies cloned couplets were cultured for 2 h to allow reprogramming to occur. Then, couplets were activated using a 2-step protocol consisting of treatment with 5 μM ionomycin for 4 to 5 min and subsequently with 2mM 6-DMAP for 4 h. Activated iSCNT embryos were cultured for 10 days inmodified SOF with various conditions (at 37 to39°C, 5 to 5.5% CO2 and 5 to 20% O2) to examine the effects ofIVC conditions. As a results, most embryos were arrested at the 8- to 16-cell stage and only 3 blastocysts were derived from rhesus monkey iSCNT. The blastocyst developmental rate was 0.26% generated from the total IVC activated interspecies embryos (n = 1153). Among the 3 blastocysts, 2 of them were used for counting nuclear number using bisbenzimide staining. The nuclear number of the 2 iSCNT-derived blastocysts was 51 and 24, respectively. The other iSCNT-derived blastocyst was used for analyzing mitochondrial (mt)DNAto confirm that it contained both cow and rhesus monkey mtDNA. As a result, mtDNA from both rhesus monkey and cow were detected inPCR analysis. The band intensity was more dominant for cow mtDNA than for rhesus monkey mtDNA. Although the blastocyst developmental rate is extremely low, it is confirmed that two phylogenetically distant species including primate could develop in vitro until the blastocyst stage by iSCNT. The in vitro developmental system of this rhesus monkey iSCNT-derived blastocysts provides a platform for further improvement of developmental rate and quality of rhesus monkey iSCNT-derived blastocysts. It also provides an opportunity to establish rhesus monkey iSCNT-derived embryonic stem cell lines for study of rhesus monkey nucleus and cow mitochondria interaction mechanisms during early developmental stages. This study was financially supported by the Korean MEST, through the BK21 program for Veterinary Science, and SNU foundation (Benefactor; RNL Bio).

Producing primate embryonic stem cells by somatic cell nuclear transfer

Nature ◽  
2007 ◽  
Vol 450 (7169) ◽  
pp. 497-502 ◽  
Author(s):  
J. A. Byrne ◽  
D. A. Pedersen ◽  
L. L. Clepper ◽  
M. Nelson ◽  
W. G. Sanger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem

scholarly journals Ultrastructural comparison of porcine putative embryonic stem cells derived by in vitro fertilization and somatic cell nuclear transfer

2016 ◽  
Vol 62 (2) ◽  
pp. 177-185 ◽  
Author(s):  
Hyunju YOO ◽  
Eunhye KIM ◽  
Seon-Ung HWANG ◽  
Junchul David YOON ◽  
Yubyeol JEON ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem

scholarly journals Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells

2017 ◽  
Vol 21 (4) ◽  
pp. 425-434
Author(s):  
Dong-Hyuk Shin ◽  
Jeoung-Eun Lee ◽  
Jin Hee Eum ◽  
Young Gie Chung ◽  
Hoon Taek Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Human Embryonic Stem Cells ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem

Effects of ovary storage temperature and embryo vitrification on somatic cell nuclear transfer outcomes in goats

2020 ◽  
Vol 32 (4) ◽  
pp. 419 ◽  
Author(s):  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Sayed Morteza Aghamiri ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mohammad Hossein Nasr Esfahani
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Storage Temperature ◽  
Storage Conditions ◽  
Farm Animals ◽  
Blastocyst Formation ◽  
Term Pregnancy ◽  
Developmental Potential ◽  
Genetic Potential

Improving the genetic potential of farm animals is one of the primary aims in the field of assisted reproduction. In this regard, somatic cell nuclear transfer (SCNT) can be used to produce a large number of embryos from genetically elite animals. The aims of the present study were to assess the effects of: (1) ovary storage conditions on preimplantation development of recovered oocytes and the freezability of the derived blastocysts; and (2) vitrification of goat SCNT-derived blastocysts on postimplantation development. Goat oocytes were recovered from ovaries and stored under warm (25°C-27°C) or cold (11°C-12°C) conditions before being used to produce SCNT embryos. There were no differences in oocytes recovered from ovaries kept under cold versus warm storage conditions in terms of cleavage (mean (±s.d.) 95.68±1.67% vs 95.91±2.93% respectively) and blastocyst formation (10.69±1.17% vs 10.94±0.9% respectively) rates. The re-expansion rate of vitrified blastocysts was significantly lower for cold- than warm-stored ovaries (66.3±8.7% vs 90±11% respectively). To assess the effects of vitrification on postimplantation development, blastocysts from cold-stored ovaries only were transferred from fresh and vitrified–warmed groups. The pregnancy rate was comparable between the fresh and vitrified–warmed groups (41.65% and 45.45% respectively). In addition, established pregnancy in Day 28-38 and full-term pregnancy rates were similar between the two groups. In conclusion, this study shows similar invitro preimplantation developmental potential of warm- and cold-stored ovaries. This study introduces the vitrification technique as an appropriate approach to preserve embryos produced by SCNT for transfer to recipient goats at a suitable time.

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