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Jinghe Liu Xingwei Liang Jiaqiao Zhu Liang Wei Yi Hou Da-Yuan Chen Qing-Yuan Sun 《遗传学报》2008,35(9):559-568
High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones. 相似文献
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Yukitoshi Takemura Motohiko Satoh Kenichi Hatanaka 《Bioscience, biotechnology, and biochemistry》2018,82(7):1159-1164
Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death. 相似文献
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Cheong HT Park KW Im GS Lai L Sun QY Day BN Prather RS 《Molecular reproduction and development》2002,61(4):488-492
The present study examined the effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of fetal fibroblast nuclear transfer (NT) porcine embryos. Frozen-thawed and serum starved fetal fibroblasts were transferred into the perivitelline space of enucleated oocytes. Cell fusion and activation were induced simultaneously with electric pulses in 0.3 M mannitol-based medium containing 0.1 or 1.0 mM CaCl(2). Some fused embryos were further activated 1 hr after the fusion treatment by exposure to an electric pulse. The NT embryos were cultured in vitro for 6 days. Fusion and blastocyst formation rates were significantly (P<0.05) increased by increasing the Ca(2+) concentration from 0.1 mM (67.1 and 6.3%) to 1.0 mM (84.7 and 15.8%). However, no difference in the number of cells in blastocysts was observed between the two groups. A higher percentage of blastocyst was also observed when control oocytes were parthenogenetically activated in the presence of elevated Ca(2+) (19.3% vs. 32.4%, P<0.05). When the reconstituted oocytes were fused in the medium containing 1.0 mM CaCl(2), increasing the number of pulses from 2 to 3 or an additional activation treatment did not enhance the blastocyst formation rate or cell number in blastocysts. These results demonstrate that increasing the Ca(2+) concentration in the fusion/activation medium can enhance the fusion and blastocyst formation rates of fetal fibroblast NT porcine embryos without an additional activation treatment. 相似文献
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Xiangpeng Dai Xiaoyang Zhao Hai Tang Jie Hao Jean-Paul Renard Qi Zhou Alice Jouneau Liu Wang 《生物学前沿》2010,5(5):439-444
The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos.
In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed
with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA
methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells
(J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem
cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not
improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either.
Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor
nuclear reprogramming. 相似文献
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Bo Fu Di Liu Hong Ma Zhen‐Hua Guo Liang Wang Zhong‐Qiu Li Fu‐Gang Peng Jing Bai 《Cell biology international》2016,40(2):214-222
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Kiho Lee Bethany K. Redel Lee Spate Jennifer Teson Alana N. Brown Kwang‐Wook Park Eric Walters Melissa Samuel Clifton N. Murphy Randall S. Prather 《Molecular reproduction and development》2013,80(2):145-154
In general, pig embryos established by somatic cell nuclear transfer (SCNT) are transferred at the one‐cell stage because of suboptimal embryo culture conditions. Improvements in embryo culture can increase the practical application of late embryo transfer. The goal of this study was to evaluate embryos cultured with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in vitro, and to track the in vivo developmental competency of SCNT‐derived blastocysts from these GM‐CSF embryos. The receptor for GM‐CSF was up‐regulated in in vitro‐produced embryos when compared to in vivo‐produced cohorts, but the level decreased when GM‐CSF was present. In vitro fertilized (IVF) embryos, supplemented with GM‐CSF (2 or 10 ng/ml), showed a higher frequency of development to the blastocyst stage compared to controls. The total cell numbers of the blastocysts also increased with supplementation of GM‐CSF. Molecular analysis demonstrates that IVF‐derived blastocysts cultured with GM‐CSF exhibit less apoptotic activity. Similarly, an increase in development to the blastocyst stage and an increase in the average total‐cell number in the blastocysts were observed when SCNT‐derived embryos were cultured with either concentration of GM‐CSF (2 or 10 ng/ml). When SCNT‐derived embryos, cultured with 10 ng/ml GM‐CSF, were transferred into six surrogates at Day 6, five of the surrogates became pregnant and delivered healthy piglets. Our findings suggest that supplementation of GM‐CSF can provide better culture conditions for IVF‐ and SCNT‐derived embryos, and pig SCNT‐derived embryos cultured with GM‐CSF in vitro can successfully produce piglets when transferred into surrogates at the blastocyst stage. Thus, it may be practical to begin performing SCNT‐derived embryo transfer at the blastocyst stage. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc. 相似文献
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体细胞核移植技术具有极其广阔的应用前景,但极低的成功率限制了这项技术在实践生产中的应用。不同的学者在不同的物种上进行了一系列的尝试,试图提高体细胞核移植的成功率。本文就体细胞核移植技术在不同物种中的成功应用进行阐述,并就如何提高体细胞核移植成功率阐明一些观点。 相似文献
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哺乳动物体细胞核移植技术在农业、生物技术、医药生产和濒危动物保护等方面具有很大的潜力和应用价值,已成为目前发育生物学研究的重要方法。但是核重编程仍是核移植技术的关键因素,制约了重构胚胎干细胞的研究。只有供核发生完全重编程,重构胚胎才能正常发育。核重编程与供核者的年龄,供核细胞的组织来源、分化状态、细胞周期、传代次数,供核细胞的表观遗传标记以及供卵者的年龄、卵子的成熟度等因素有关。创造各种适于核重编程的条件有利于从更高的起点开展核移植胚胎干细胞的研究,提高重枸胚胎干细胞建系效率。 相似文献
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In this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6% compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected (0.9%) without any live fetuses and offspring. 相似文献
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Bethany R. Mordhorst Joshua A. Benne Raissa F. Cecil Kristin M. Whitworth Melissa S. Samuel Lee D. Spate Clifton N. Murphy Kevin D. Wells Jonathan A. Green Randall S. Prather 《Molecular reproduction and development》2019,86(5):558-565
Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1–3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere‐like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer. 相似文献
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细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。 相似文献
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Stem cell research in China 总被引：2，自引：0，他引：2
Liao L Li L Zhao RC 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2007,362(1482):1107-1112
In the past 5 years, China has increased its efforts in the field of stem cell research and practice. Basic research mainly focuses on bone marrow and embryonic stem cells. Clinical applications of stem cells in the treatment of acute heart failure, acute liver failure and lower limb ischaemia have been reported by many hospitals. China enacted its 'Ethical Guidelines for Human Embryonic Stem Cell Research' in 2003. At present, China has the most liberal and favourable environments for human embryonic stem cell research. 相似文献