Analysis of Nuclear Reprogramming Following Nuclear Transfer to Xenopus Oocyte

2014 ◽  
pp. 71-82 ◽  
Author(s):  
Jerome Jullien
Keyword(s):  
Nuclear Transfer ◽  
Xenopus Oocyte ◽  
Nuclear Reprogramming

scholarly journals Technical Challenges in the Derivation of Human Pluripotent Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Parinya Noisa ◽  
Rangsun Parnpai
Keyword(s):  
Stem Cells ◽  
Cell Biology ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Stem Cell Biology ◽  
Nuclear Reprogramming ◽  
Pluripotent Cells ◽  
Ethical Concerns ◽  
Technical Challenges

It has long been discovered that human pluripotent cells could be isolated from the blastocyst state of embryos and called human embryonic stem cells (ESCs). These cells can be adapted and propagated indefinitely in culture in an undifferentiated manner as well as differentiated into cell representing the three major germ layers: endoderm, mesoderm, and ectoderm. However, the derivation of human pluripotent cells from donated embryos is limited and restricted by ethical concerns. Therefore, various approaches have been explored and proved their success. Human pluripotent cells can also be derived experimentally by the nuclear reprogramming of somatic cells. These techniques include somatic cell nuclear transfer (SCNT), cell fusion and overexpression of pluripotent genes. In this paper, we discuss the technical challenges of these approaches for nuclear reprogramming, involving their advantages and limitations. We will also highlight the possible applications of these techniques in the study of stem cell biology.

scholarly journals Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis

Cell Research ◽  
2007 ◽  
Vol 17 (2) ◽  
pp. 135-150 ◽  
Author(s):  
Tianlong Gao ◽  
Junke Zheng ◽  
Fengying Xing ◽  
Haiyan Fang ◽  
Feng Sun ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Normal Development ◽  
Nuclear Reprogramming

scholarly journals Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

2017 ◽  
Author(s):  
◽  
Bethany Rae Mordhorst
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Pharmaceutical Agents ◽  
Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

Nuclear reprogramming by somatic cell nuclear transfer – the cattle story

2007 ◽  
Vol 6 (1) ◽  
pp. 327-339
Author(s):  
XC Tian ◽  
SL Smith ◽  
SQ Zhang ◽  
C Kubota ◽  
C Curchoe ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Nuclear Reprogramming

Antioxidant treatment during manipulation procedures prevents mitochondrial and DNA damage and enhances nuclear reprogramming of bovine somatic cell nuclear transfer embryos

2015 ◽  
Vol 27 (7) ◽  
pp. 1088 ◽  
Author(s):  
Hyo-Kyung Bae ◽  
In-Sun Hwang ◽  
Ji-Ye Kim ◽  
Sung-Young Lee ◽  
Choon-Keun Park ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Damage ◽  
Membrane Potential ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Nuclear Reprogramming ◽  
Antioxidant Treatment ◽  
Treatment Groups ◽  
H2o2 Level

We tried to prevent the mitochondrial and DNA damage caused by mechanical stress-associated reactive oxygen species (ROS), and to improve the reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos by antioxidant treatment during the manipulation procedures of SCNT. Bovine recipient oocytes and reconstituted oocytes were treated with antioxidants during manipulation procedures. The H2O2 level, mitochondrial morphology, membrane potential and apoptosis at the one-cell stage, and in vitro development and DNA methylation status of blastocysts were evaluated. Antioxidant treatment during manipulation procedures reduced the H2O2 level of SCNT embryos. Antioxidant-treated SCNT embryos normally formed mitochondrial clumps, similar to IVF embryos, and showed higher mitochondrial membrane potential versus the SCNT control (P < 0.05). Apoptosis and DNA fragmentation were reduced by antioxidant treatment. The development rate to the blastocyst stage was higher (P < 0.05) in the antioxidant treatment groups (30.5 ± 2.5 to 30.6 ± 1.6%) versus the control (23.0 ± 1.9%). The DNA methylation status of blastocysts in the antioxidant treatment groups was lower (P < 0.05) than that of the control and similar to that of IVF embryos. These results indicate that antioxidant treatment during manipulation procedures can prevent cellular damage that may be caused by mechanical stress-associated ROS, and improve nuclear reprogramming.

scholarly journals Coexpression analysis identifies nuclear reprogramming barriers of somatic cell nuclear transfer embryos

Oncotarget ◽  
2017 ◽  
Vol 8 (39) ◽  
pp. 65847-65859 ◽  
Author(s):  
Yongchun Zuo ◽  
Guanghua Su ◽  
Lei Cheng ◽  
Kun Liu ◽  
Yu Feng ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Nuclear Reprogramming ◽  
Coexpression Analysis

65 NUCLEAR REPROGRAMMING OF PORCINE CELLS AND THEIR USE AS DONOR CELL FOR NUCLEAR TRANSFER AFTER TREATMENT IN XENOPUS EGG EXTRACTS

2007 ◽  
Vol 19 (1) ◽  
pp. 150 ◽  
Author(s):  
K. Miyamoto ◽  
M. Ohnuki ◽  
N. Minami ◽  
M. Yamada ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Nuclear Reprogramming ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Revealing an adequate cell state for nuclear reprogramming is essential to achieve efficient production of cloned embryos and animals. Previous reports suggest that nuclei from undifferentiated cells such as blastomeres or embryonic stem cells can support efficient development of cloned embryos to term. In recent years, differentiated somatic cells are frequently used for donor cells because of ease of preparation and application for genetic modification. The efficiency of the somatic cell nuclear transfer (SCNT) is still extremely low. We hypothesized that somatic cells that had been reprogrammed to dedifferentiated states before SCNT might support higher developmental ability of SCNT embryos. To test this hypothesis, porcine fibroblast cells were treated with Xenopus egg extracts, and the extract-treated cells (ETCs) were used as donor cell for SCNT to examine their ability to support early embryonic development. Xenopus egg extracts were prepared from activated S-phase eggs. Porcine fibroblast cells (106/mL) were permeabilized by 500 ng mL-1 of Streptolysin O and were incubated in the egg extracts with the energy-regenerating system for 2 hours at 23�C. After the extract treatment, permeabilized membranes were resealed in DMEM containing 2 mM CaCl2. The ETCs were fused with porcine enucleated oocytes and simultaneously activated. The reconstructed embryos were cultured in PZM-3 medium for 7 days. All statistical differences were analyzed by ANOVA. Reprogramming of ETCs was evaluated on changes of chromatin states and gene expression. Chromatin-binding proteins of ETCs were separated and analyzed on SDS-PAGE. Some proteins were incorporated onto and/or released from chromatins after the extract treatment. Especially, Xenopus egg-specific linker histone B4 was assembled on chromatins. Non-permeabilized control cells did not show these protein exchanges. Deacetylation of histone H3 lysine9 was detected in half number of ETCs in an ATP-dependent manner. In contrast, a high population of histone H3-acetylated cells was observed in buffer-treated cells as well as cells before the extract treatment. The pluripotent marker gene expression, such as OCT4 and SOX2, was also observed in ETCs after culture. The gene expression of these genes was not detected in non-treated cells. These results indicate that the extract treatment induces or triggers a part of dedifferentiation of somatic cells. These ETCs were used as donor cell for SCNT, and reconstructed cloned embryos were cultured. SCNT embryos showed no significant difference in cleavage rates and developmental rates to the blastocyst stage (25%) compared with non-treated control cells (26%). However, the total cell number of embryos at the blastocyst stage was significantly higher in SCNT embryos from ETCs compared with those of control cells (62 � 7 vs. 43 � 2, respectively; P &lt; 0.05). These results indicate that the extract treatment before nuclear transfer may stimulate cell proliferation of SCNT embryos but not improve early development. More studies, however, are needed to investigate their developmental ability to term.

Scriptaid Improves In Vitro Development and Nuclear Reprogramming of Somatic Cell Nuclear Transfer Bovine Embryos

2011 ◽  
Vol 13 (5) ◽  
pp. 431-439 ◽  
Author(s):  
Li-Jun Wang ◽  
Hui Zhang ◽  
Yong-Sheng Wang ◽  
Wen-Bing Xu ◽  
Xian-Rong Xiong ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Nuclear Reprogramming ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development
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