共查询到20条相似文献,搜索用时 15 毫秒
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In-Sun Hwang Hyo-Kyung Bae Hee-Tae Cheong 《Journal of veterinary science (Suw?n-si, Korea)》2013,14(3):235-240
The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels. 相似文献
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Goo Jang So Gun Hong Byeong Chun Lee 《Journal of veterinary science (Suw?n-si, Korea)》2011,12(1):83-89
Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures. 相似文献
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Comparison of efficiency of in vitro cloned sheep embryo production by conventional somatic cell nuclear transfer and handmade cloning technique 下载免费PDF全文
S Khan M Tali A Khan S Bhat A Ashraf MH Bhat F Khan RA Shah 《Reproduction in domestic animals》2018,53(2):512-518
Conventional somatic cell nuclear transfer (SCNT) technique of in vitro production of cloned embryos involves use of costly and complicated micromanipulators. Handmade cloning (HMC) technique has been applied as efficient and cost‐effective alternative in many livestock species. The aim of the present study was to compare the efficiency of in vitro production and in vitro development of cloned sheep embryos by the two techniques. Cloned embryos were produced by conventional SCNT using micromanipulator apparatus and by HMC technique. Enucleation efficiency and efficiency of fusion with somatic cell (nucleus donor) were compared. Cleavage percentage was observed on day 2 of in vitro culture (IVC), and morula and blastocyst percentages were calculated on day 7 of IVC. Higher enucleation efficiency (96.98 ± 1.01 vs. 93.62 ± 1.03; p > .05) as well as fusion efficiency was obtained with HMC technique than with conventional SCNT (96.26 ± 1.34 vs. 92.63 ± 0.70, p < .05); 181 cloned sheep embryos were produced in vitro by conventional SCNT and 92 by HMC. Cleavage percentage observed on day 2 of in vitro culture was higher in HMC than SCNT (66.92 ± 3.72 vs. 55.97 ± 2.5, respectively, p < .05). Morula percentage obtained was higher in SCNT than HMC (44.12 ± 2.93 vs. 30.43 ± 6.79, respectively, p < .05), whereas blastocyst percentage obtained by HMC was higher (12.46 ± 4.96) than SCNT (5.31 ± 2.25; p > .05). It was inferred that HMC technique provides a cost‐effective and efficient method of in vitro production of cloned sheep embryos with a comparatively simpler technique with a possibility of automation. Efficiency of cloned embryo production could be improved further by propagating and standardizing this technique. 相似文献
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Yu-Ting Zhang Wang Yao Meng-Jia Chai Wen-Jing Liu Yan Liu Zhong-Hua Liu Xiao-Gang Weng 《Journal of veterinary science (Suw?n-si, Korea)》2022,23(2)
BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos. 相似文献
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Min Jung Kim Geon A Kim Young Kwang Jo Yoo Bin Choi Byeong Chun Lee 《Animal Science Journal》2016,87(1):21-26
The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies. 相似文献
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牛体细胞核移植胚胎的批量化生产 总被引:8,自引:0,他引:8
利用2枚显微去核后的半卵与1枚供体细胞同步融合,并结合微穴法(WOWs)培养体系批量化生产成年牛克隆胚胎(日产40~80枚).结果,重构胚电融合率95.7%(3059/3197)、卵裂率87.1%(2637/3027)、囊胚率41.1%(1244/3027)和可冻胚率(72.5%,933/1244)均达到较高水平.克隆胚胎采用玻璃微管玻璃化冷冻保存数月后移植,30日龄时妊娠率为28.1%(48/171),已经产下5头足月克隆牛续.结果表明,该方法具有产业开发潜力. 相似文献
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为了检测猪体细胞核移植囊胚的质量和全能性基因表达量,采用体细胞核移植技术制备克隆胚胎并于体外培养5 d后获得囊胚,对照组为从人工授精5 d后的长白母猪体内获取的体内囊胚。在高倍镜下检测两组囊胚的形态,用Hoechest 33342染色细胞核DNA,记录囊胚细胞总数;建立单胚胎cDNA的制备方法,并用qPCR检测囊胚中全能性基因(Oct4、Nanog、Sox2)的表达量。结果表明,与体内囊胚相比,猪体细胞核移植囊胚质量较差,细胞数目显著降低(分别为110±10.3和54±12.6);并且全能性基因表达量显著下降(P<0.05)。由此可见,全能性基因表达量偏低是影响猪体细胞核移植囊胚发育能力的因素之一。 相似文献
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Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development 下载免费PDF全文
W‐J Lee J‐H Lee R‐H Jeon S‐J Jang S‐C Lee J‐S Park S‐L Lee W‐A King G‐J Rho 《Reproduction in domestic animals》2017,52(3):437-445
Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm‐transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro‐fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts. 相似文献
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To obtain data concerning the survival of embryos and calves derived from somatic cell nuclear transfer (SCNT) in Japan, a nationwide survey was carried out in April, 2009. As a result, data concerning 3264 embryo transfers (ETs) with SCNT embryos which produced 301 calves were accumulated and their survival was analyzed. The present survey revealed that survival rates of transferred bovine embryos and produced calves derived from SCNT had not improved over a decade (1998–2007). A remarkable feature of the pregnancies with SCNT embryos was a high incidence of spontaneous abortions. When the decade was divided by the occurrence of bovine spongiform encephalopathy (BSE) in 2001, significant decreases in the ‘after BSE’ period (2002–2007) were observed in the percentages of calves born (P < 0.01), calves living at birth (P < 0.05), calves living for 24 h (P < 0.05) and 6 months (P < 0.01). Abortions that occurred during 61–99 days after ETs were significantly increased (P < 0.01) in the ‘after BSE’ period. Certain kinds of regeneration that occurred in oocytes during the 15–20 h of storage of bovine ovaries at 10–15°C as a part of BSE inspection might have had some negative effects on SCNT embryos when these oocytes were used as recipients of SCNT. 相似文献
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Xiao-ying He Li-bing Ma Xiao-ning He Wan-tong Si Yue-Mao Zheng 《Journal of veterinary science (Suw?n-si, Korea)》2016,17(2):145-152
Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. 相似文献
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Katsuhiro OHKOSHI Seiya TAKAHASHI Shin-ichiro KOYAMA Satoshi AKAGI Noritaka ADACHI Tadashi FURUSAWA Jun-ichiro FUJIMOTO Yoshiaki IZAIKE Tomoyuki TOKUNAGA 《Animal Science Journal》2003,74(4):269-276
In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO2, 5% O2, 90% N2 for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%). 相似文献
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This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or β-mercaptoethanol (β-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 µM CYS or 100 µM β-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. β-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos. 相似文献
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Ting-Yu ZHANG Jian-Jun DAI Cai-Feng WU Xiao-Long GU Liang LIU Zhi-Qiang WU Yi- Ni XIE Bin WU Hui-Lan CHEN Yao LI Xue-Jin CHEN De-Fu ZHANG 《Animal Science Journal》2012,83(2):103-110
To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs. 相似文献
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体细胞核移植(somatic cell nuclear transfer,SCNT)是一种能将已分化的体细胞重编程为全能胚胎的繁殖生物技术,在良种扩繁、濒危物种保护和治疗性克隆等方面有着广泛的应用前景,但极低的克隆效率、克隆动物胎盘异常、出生后胎儿畸形等严重限制了该技术的实际应用。造成克隆效率低和胚胎发育异常的主要原因是供体核表观遗传重编程错误或不完全。1958年,将非洲爪蟾(Xenopus laevis)幼体肠细胞核移入去核卵母细胞,获得了第1例SCNT动物个体;1986年,通过电融合1个卵裂球与去核卵母细胞成功获得了3只存活的羔羊;1997年,将成年母羊的乳腺上皮细胞与去核卵细胞电融合,获得首个SCNT哺乳动物"多利",开启了克隆时代,目前牛、小鼠、山羊、猪、欧洲盘羊、家兔、家猫、马、大鼠、骡子、狗、雪貂、狼、水牛、红鹿、单峰骆驼、食蟹猴等相继成功克隆,其中最引人瞩目的是2018年食蟹猴的成功克隆。作者通过将SCNT胚胎与受精胚胎的发育进行对比,阐述了SCNT过程中DNA甲基化、组蛋白修饰、基因组印迹、染色体状态等的重编程过程和缺陷,并从表观修饰剂、组蛋白去甲基化酶、抑制Xist表达、补充鱼精蛋白和精子RNA方面探讨单独或联合消除表观遗传重编程障碍对克隆效率的影响。随着低样本量测序技术的发展和完善,人们能够在SCNT胚胎中检测到更详细的全基因组表观遗传修饰图谱,进一步揭示SCNT胚胎表观遗传重编程中的缺陷,为提高克隆效率提供了线索。通过上述内容的阐述,希望为后续开发联合消除多种表观遗传障碍而提高克隆效率的策略和思路。 相似文献