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1.
This study was designed to examine the ability of rabbit metaphase II oocyte cytoplasm to support the development of interspecies nuclear transfer embryos reconstructed using donor nuclei from different species. Skin fibroblast cells from a camel and Tibetan antelope were used as donor nuclei. As a first step, we investigated the efficiency of different activation protocols by comparing the parthenogenetic development of rabbit oocytes. The protocol that yielded the highest blastocyst rate was used to activate the reconstructed embryos in nuclear transfer experiments. In addition, the effect of donor cell serum starvation on the development of the reconstructed embryo was also examined. More than half of the karyoplast-cytoplast couplets could be fused, and about one third of the reconstructed embryos were capable of completing first cleavage, regardless of the species of donor nuclei. Some of the cleaving reconstructed embryos were even capable of progressing further and developing to the blastocyst stage (1.4-8.7% for the Tibetan antelope and 0-7.5% for the camel, respectively). Our results suggest that the mechanisms regulating early embryo development may be conserved among mammalian species and some factors existing in rabbit oocyte cytoplasm for somatic nucleus reprogramming and dedifferentiation may not be species-specific. Rabbit oocyte cytoplasm can reprogram donor nuclei regardless of the origin of the nucleus and support in vitro development to an advanced stage.  相似文献   

2.
Interspecies cloning may be a useful method to help conserve endangered species and to study nuclear-cytoplasm interaction. The present study investigated in vitro development of goral (Naemorhedus goral) intergeneric nuclear transfer embryos produced by fusing goral fibroblasts with enucleated metaphase II (MII) bovine oocytes. After two to five passages, serum-starved or non-starved goral skin fibroblast cells were transferred into enucleated MII bovine oocytes. Couplets were electrically fused and chemically activated, and then cultured in either modified synthetic oviduct fluid (mSOF) or tissue culture medium-199 (TCM-199) supplemented with 10% FBS. Serum starvation of donor cells did not affect the fusion rate and or development to of cells to the two-cell stage, to more than 9-cells, or to morulae, regardless of culture medium. Three blastocysts from 202 fused embryos were obtained when embryos reconstructed with non- serum- starved donor cells were cultured in mSOF. However, no blastocysts were obtained when the embryos reconstructed with serum-starved donor cells were cultured in mSOF. The total cell number of goral intergeneric embryos averaged 130.3 (range 105-180). In conclusion, this study demonstrated that bovine oocytes can support blastocyst development after intergeneric SCNT with goral fibroblasts.  相似文献   

3.
Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear–mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8‐cell stage (P > 0.05). However, buffalo iSCNT embryos did not develop beyond the 16‐cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8‐ to 16‐cell stage (P > 0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P < 0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8–16‐cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear–mitochondrial interactions.  相似文献   

4.
The possibility of producing HanWoo (Bos taurus coreanae) calves from transferable bovine embryos, obtained by interbreed nuclear transfer using Holstein cytoplasts and surrogates, was investigated. As donor nuclei, HanWoo fetal fibroblasts were used. Cells were induced into quiescence by serum deprivation for 4-7 days before nuclear transfer. In vitro matured Holstein oocytes were enucleated, and single donor cell was placed into the perivitelline space of enucleated oocyte. After reconstruction, the embryos were fused. activated and cultured. On day 7, the embryos that developed to the blastocyst stages were transferred into Holstein recipient cows on day 6 to 7 of estrous cycle (estrus=0). The reconstructed embryos were successfully fused (58.8%; 47/80), cleaved (91.5%; 43/47), and developed to blastocysts (29.8%; 14/47). Eleven blastocysts were transferred into 5 Holstein recipient cows. Two recipients were pregnant, confirmed by ultrasonography at day 60 of gestation. But, one of them was opened between on day 80 to 100 of pregnancy, and the other had a stillbirth on day 255. The stillborn calf was physically normal, and we couldn't find any evidence of anomaly. These results show that cells derived from HanWoo somatic cell lines can be reprogrammed by interbreed nuclear transfer and develop subsequently in vivo as well as in vitro.  相似文献   

5.
The effects of Ca(2+) concentration in activation medium and cytochalasin B treatment after activation on the parthenogenetic development of pig oocytes were examined. In addition, cloned embryos derived from miniature pig somatic cells were activated under optimal conditions and the effects of Ca(2+) in fusion medium on the development of embryos after activation was examined. When oocytes were activated in 0.1 mM Ca(2+) and then treated with cytochalasin B, the blastocyst formation rate (28.6%) was significantly higher than those activated in 0-0.05 or 1.0 mM (11.0-18.3%). Treatment with cytochalasin B decreased the second polar body extrusion rate of activated oocytes. The presence or absence of Ca(2+) in fusion medium did not affect the fusion rate of miniature pig somatic cells with recipient oocytes. A few cloned embryos developed to the blastocyst stage (2.7-9.0%) without an additional activation treatment. On the other hand, significantly more embryos developed to the blastocyst stage after activation treatment when they were fused in the absence (28.9%) of Ca(2+) rather than the presence (16.5%) of it. These results show that the highest blastocyst formation rate for miniature pig cloned embryos is obtained when donor cells and recipient oocytes are fused in the absence of Ca(2+) and then activated in 0.1 mM Ca(2+) and treated with cytochalasin B.  相似文献   

6.
This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.  相似文献   

7.
本研究检测人神经母细胞瘤细胞-水牛和人神经母细胞瘤细胞-猪重构胚胎的体外发育潜能,以期建立人神经细胞母细胞瘤细胞的异种治疗性克隆模型,为进一步研究人癌症发生过程中的表观遗传学修饰机制及分离得到正常的人类胚胎干细胞奠定基础。分别以水牛颗粒细胞和人神经母细胞瘤细胞为供体,水牛卵母细胞为受体构建克隆胚胎,然后检测这2种重构胚胎的体外发育效率;再以猪卵母细胞为胞质受体,同样以上述2种体细胞为供体细胞构建重构胚胎。结果显示,人神经母细胞瘤细胞-水牛重构胚胎囊胚发育率显著低于水牛同种克隆胚胎的（1.1%∶12.1%,P〈0.05）,而融合率（83.7%∶79.4%,P〉0.05）和卵裂率（55.8%∶58.2%,P〉0.05）无显著差异。但是,当以猪卵母细胞为受体胞质时,人-猪重构胚胎卵裂率显著低于水牛-猪重构胚胎（43.7%∶89.8%,P〈0.05）,但重构胚胎融合率和囊胚率差异不显著（85.8%∶82.5%,0∶1.4%,P〉0.05）。结果表明,人神经母细胞瘤细胞-水牛和人神经母细胞瘤细胞-猪异种重构胚胎可以在体外发育,尽管其囊胚发育率相对比较低。  相似文献   

8.
This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.  相似文献   

9.
The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -35 degrees C or -80 degrees C in the presence of 5% DMSO were used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstructed from the somatic cells, 78% to 81% showed cleavage, 43% to 48% reached the stage of morula formation and 34% to 35% reached blastocyst formation. There were no significant differences in development (P>0.05) when the NT embryos were compared with those reconstructed from fresh somatic-cell-derived skin tissues (75%, 45%, and 38%, for cleavage, and development to morula and blastocyst stages, respectively). The results indicated that cell lines derived from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer. The resulting embryos showed similar development in vitro to those reconstructed from unfrozen fetal-skin-derived somatic cells.  相似文献   

10.
试验采用脂质体转染法与电穿孔法,以携带绿色荧光蛋白（GFP）-新霉抗性（neo-）双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF（表皮生长因子）对转基因胚的影响,不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示,体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高,但对后期转基因重构胚的囊胚发育率的影响,以添加EGF 20 ng/mL组的最高;以胎牛耳成纤维细胞为供体细胞,不同转染方法转染供体细胞构建重构胚,其囊胚发育率差异不显著（P>〖JP2〗0.05）;mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好,该体系更适合体细胞核移植法生产转基因牛胚胎。  相似文献   

11.
小鼠卵母细胞化学去核及手工构建核移植胚胎   总被引：1，自引：1，他引：0  
为优化脱羰秋水仙碱（DC）诱导去核程序,研究以DC去核卵母细胞为核受体的、无透明带体细胞核移植方法在小鼠体细胞核移植中的应用,试验比较了乙醇、SrCl2两种激活方法及脱羰秋水仙碱处理开始时间对小鼠MⅡ期卵母细胞去核效率的影响;将DC诱导去核成功的卵母细胞去除透明带,与胎儿成纤维细胞粘合、电融合和SrCl2激活后,体外培养重构胚。结果显示,7%乙醇激活后0 min起始DC处理可得到最高的诱导去核率（66.4%）;而在8 mmol/L SrCl2中激活15 min后用DC处理可得到最高的诱导去核率（64.3%）;目前重构胚可以体外发育到8-细胞。试验结果首次证明了SrCl2在小鼠卵母细胞DC诱导去核中的作用效果与乙醇相当,初步证明了将DC诱导去核技术与无透明带技术相结合手工克隆生产小鼠重构胚的可能性,它的成功将大大简化核移植程序。  相似文献   

12.
研究旨在探讨猪卵母细胞核因子在重编程过程中发挥的作用。将体细胞引入未去核的MⅡ期卵母细胞中,构建体细胞核与卵母细胞核共存的核移植四倍体胚胎。通过分析核移植四倍体胚胎的早期发育情况探讨卵母细胞核因子对核移植四倍体胚胎早期发育的影响。结果显示,核移植四倍体胚胎、孤雌二倍体胚胎及孤雌单倍体胚胎这3组胚胎的卵裂率极显著高于核移植二倍体胚胎（P<0.01）,且核移植四倍体囊胚率及总细胞数也极显著高于核移植二倍体囊胚（P<0.01）。与通过标准核移植程序构建的核移植二倍体胚胎相比,核移植四倍体胚胎具有更强的发育能力。本研究建立了一个体细胞核与完整卵母细胞核因子物质共存的四倍体胚胎模型,有助于研究供体核与卵母细胞核之间的联系,为研究核因子在重编程过程中发挥的作用提供了平台。  相似文献   

13.
The study was aimed to investigate the role of porcine oocyte nuclear factors during reprogramming. Somatic cell nuclei was introduced into intact MⅡ oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. And then the influence of the oocyte nucleus on tetraploid SCNT embryo development was examined by assessing characteristics including cleavage rate and blastocyst rate. The results showed that the cleavage rate of tetraploid SCNT embryos,diploid parthenogenetic embryos and haploid parthenogenetic embryos was extremely significantly higher than that of standard diploid SCNT embryos (P<0.01). The blastocyst rate and the total number of cells in tetraploid SCNT embryos were extremely significantly higher than that of standard diploid SCNT embryos (P<0.01).Overall,tetraploid SCNT embryos had a higher developmental competence than standard diploid SCNT embryos. In conclusion, the embryonic model was established in which a fetal fibroblast nucleus and an oocyte M Ⅱ plate coexist. Tetraploid SCNT represented a new research platform that was potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.  相似文献   

14.
本研究探讨了核移植前对受体卵子进行激活、细胞融合开始时间及供体受精卵细胞周期调节对核移植卵体外发育的影响。其结果显示核移植前对受体卵子激活组的细胞融合率与对照组没有差异 ,但重组胚胎的卵裂率、8～ 16细胞期胚胎及囊胚的发育率比对照组明显提高 ;核移植前激活的受体卵子分别在卵子体外成熟开始的第30h和 4 5h与供体细胞进行细胞融合 ,结果 ,30h组的细胞融合率和卵裂率与 4 5h组没有差异 ,但发育到 8～ 16细胞期及囊胚的发育率均比 4 5h的高 ;将供体受精卵用诺考达唑 (Nocodazole)处理后 ,进行核移植的结果 ,处理组的细胞融合率、卵裂率、发育到 8～ 16细胞期和囊胚的发育率与对照组无差异  相似文献   

15.
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16.
以体外培养的鲁西黄牛耳皮肤成纤维细胞作核供体，研究利用体细胞克隆技术保存我国地方黄牛优良品种的技术方法。通过组织块培养法建立的鲁西黄牛（1♂，4♀）成纤维细胞系，作为核移植供体细胞，利用屠宰场母牛卵巢卵母细胞经体外培养成熟后作为核受体进行核移植，试验结果表明：重构胚的融合率为62．5％（242／387），分裂率为63．6％（154／242），体外培养第7天囊胚发育率为42．9％（66／154）。体外培养第7天的囊胚的内细胞团细胞（ICM）与滋养层细胞数平均为37和47，ICM占44．2％。体外发育到7d的囊胚新鲜胚胎的移植妊娠率（新鲜胚胎）移植受体10头，60d妊娠率为20％（2／10）。  相似文献   

17.
The objective of the study was to investigate interspecies Somatic Cell Nuclear Transfer (iSCNT) techniques in marbled cats (Pardofelis marmorata), using domestic cat and rabbit oocytes as the recipient cytoplasm. The recipient oocytes were obtained from ovariohysterectomized cats and superovulated rabbits. The donor cells were collected from a male marbled cat that had died in captivity. Experiment 1 was conducted to observe the development of cloned marbled cat embryos (marbled cat donor cells-domestic cat oocytes; MC-DC), derived from oocytes matured for 24, 36 and 42 h. The result showed that the developmental rates of MC-DC cloned embryos at the 4-8 cell and the morula stages derived from oocytes cultured for 24 h were significantly greater than those cultured for 36 and 42 h (p < 0.05). Experiment 2 was conducted to compare the fusion rate of MC-DC couplets, fused by inducing different fusion voltages, 2.1 or 2.4 kV/cm. The result showed that there was no difference in fusion efficiency between the 2.1 and 2.4 kV/cm fusion protocols. Experiment 3 was conducted to compare the developmental rate of MC-DC and domestic cat (DC-DC) cloned embryos. In vitro fertilized cat embryos served as a control. The development of MC-DC and DC-DC cloned embryos to the 4- to 8-cell, morula and blastocyst stages was not significantly different. However, the development rates at morula and blastocyst stages of control were significantly greater than those of cloned embryos (p < 0.05). Experiment 4 rabbit (RB) oocytes were used as a recipient cytoplasm for marbled cat and domestic cat cloned embryos (MC- RB and DC-RB). RB-RB cloned embryos served as a control. There were no differences in the developmental rates between MC-RB, DC-RB and RB-RB embryos. In conclusion, marbled cat fibroblast cells can be reprogrammed in domestic cat and rabbit oocytes, and by using iSCNT it might be possible to produce marbled cat offspring in the future.  相似文献   

18.
利用脂质体转染法获得转Toll样受体4(Toll-like receptors 4,TLR4)基因的成纤维细胞,将其作为供体细胞,再通过体细胞核移植技术构建重构胚,最终生产出转TLR4基因且性别可控的大尾寒羊。本研究通过原代培养大尾寒羊胎儿皮肤成纤维细胞,并进行SRY基因性别鉴定,利用脂质体转染法转入TLR4基因,然后分别从形态学与分子水平检测TLR4基因的表达,将稳定表达转TLR4的成纤维细胞核移植入绵羊去核卵母细胞中构建重构胚。最终获得5个稳定表达TLR4的阳性细胞克隆,经SRY基因性别鉴定为雌性细胞株,通过实时荧光定量PCR分析,在第4代转染的细胞中TLR4表达量最高,比未转染细胞高出7.4816倍(P<0.01),卵裂的重构胚中有EGFP的表达。试验成功构建出含TLR4基因的重构胚,为今后生产出性别可控的抗病转基因绵羊地方新品种奠定基础。  相似文献   

19.
Exploiting the method of liposome transfection to obtain modified Toll-like receptors 4(TLR4) gene fibroblast cells as competent donor, build reconstructed embryos, which finally produced transfected TLR4 gene and gender controllable Large-tailed Han sheep by somatic cell nuclear transfer technology.In this study, through primary culturing the fetus dermal fibroblasts from Large-tailed Han sheep, to identify the SRY gene gender.Using the method of liposome transfection transferred TLR4 gene into fibroblast cells, then detected whether TLR4 gene expression stably or not in morphology level and molecular level respectively, transplanting fibroblast cells which stably expressed TLR4 gene into oocytes without nucleus from Large-tailed Han sheep for building reconstructed embryos.To harvest 5 positive clones by fibroblast cells with stable expression level of TLR4.According to the identification of the SRY gene gender, the cell lines were female.The analysis of Real-time fluorescent quantitative PCR, the fourth generation of transfected cells had the highest expression level of TLR4, which were higher than control group in 7.4816 times (P<0.01).The cleavage reconstructed embryos had stable expression level of EGFP.We succeeded in building reconstructed embryos with TLR4 gene and lay the foundation for producing the disease-resistant and gender controllable transgenic sheep in the future.  相似文献   

20.
In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.  相似文献   

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