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兔核移植胚胎的克隆研究 总被引：12，自引：0，他引：12
本研究改进了兔受体卵母细胞的去核程序及供体卵裂球的处理方法，并对核移植胚的体外克隆、继代克隆及克隆胚的体内发育进行了试验。结果表明：卵龄对受体卵母细胞的去核具有显著的影响，卵龄为１５～１８ｈ的去核率为１００％（４５／４５），显著高于１３～１４ｈ的４６．６％（７／１５），Ｐ＜０．０１。ＤＮＡ合成抑制剂Ａｐｈｉｄｉｃｏｌｉｎ处理１６－细胞期卵裂球后，重组胚的囊胚发育率５４％（２０／３７）高于未处理组的４５％（１４／３１），Ｐ＜０．０５。从２枚１６－细胞供体胚分别获得来自一个供体胚的９枚和７枚核移植克隆囊胚，囊胚发育率为５１．６（１６／３１）。然而第一次继代核移植胚的囊胚发育率仅为１１．５％（６／５２）。１６－及３２－细胞期卵裂球的重组胚可发育至产仔，共获２窝８只仔兔。其中１只、２只、２只和３只仔兔分别来自４个供体胚。 相似文献
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采用核移植技术将单个细胞注入去核2-细胞卵裂球中,比较来自于囊胚的内细胞团(ICM)和滋胚层细胞(TE)产生孕体或嵌合体的发育潜力。用TE和ICM重构胚胎的发育潜力,主要取决于注射hCG后从输卵管收集到的2-细胞胚胎的时间。在注射hCG后38～42 h和43～46 h收集得到的2-细胞胚胎,其重构胚胎仅仅能够发育到4-细胞期。注射hCG后48～51 h收集得到的2-细胞胚胎,重构后发生卵裂的胚胎比率显著提高,有少部分会发育到囊胚期。用诺考达唑(Nocodazole)处理这些重构胚,发生卵裂的胚胎比率会显著提高,并且有部分重构胚发育到囊胚阶段。将这些囊胚移植到受体鼠中,在其妊娠中期未检测到供体核的出现。用ICM和TE进行重构得到的嵌合2-细胞胚胎,其体外发育潜力有限,本试验中也未得到嵌合孕体。 相似文献
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旨在探讨MG-132对牛体细胞核移植重构胚重编程的影响。将牛卵母细胞体外培养至MI期,添加不同浓度的MG-132(0、3、5、7、10μmol·mL-1),至成熟后进行激活,通过胚胎的发育情况得出MG-132的合适浓度,并用此浓度的MG-132作用于核移植全过程,来研究MG-132的作用,用免疫组化的方法进一步验证结果。结果,5、7μmol·mL-1MG-132组卵裂率和囊胚率均显著低于对照组和2μmol·mL-1组(P0.05),但这2组间无显著差异(P0.05),而10μmol·mL-1组卵裂率只有23.44%,低于其他各组(P0.05);成熟卵母细胞在5μmol·mL-1MG-132中分别作用0、2、4h激活后,卵裂率、囊胚率均无显著差异(P0.05),而6h组激活后卵裂率、囊胚率均明显降低(P0.05);核移植操作过程中添加MG-132组的卵裂率与对照组相比无显著差异(P0.05),而桑葚胚和囊胚率都较对照组显著升高(P0.05);未添加MG-132组,融合后1h供体细胞几乎无变化,4h后核纤层蛋白(lamin)着色显著,说明染色体凝集不完全;添加组融合4h后,核膜不完全着色,明显发生了染色体凝集。上述结果说明5μmol·mL-1MG-132能够促进重构胚的重编程,但对重构胚移植后的附植和妊娠作用还有待进一步研究。 相似文献
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供体细胞的不同处理方法对牛核移植重构胚发育能力的影响 总被引：1，自引：0，他引：1
为了研究供体细胞不同的处理方法对核移植重构胚的作用，比较了用于保种的冻存和新鲜的成纤维细胞做供体细胞、供体细胞不同的离心转数、供体细胞不同的血清饥饿时间及用本实验室冻存的成纤维细胞，解冻复苏后，不同传代次数对重构胚的影响。结果表明分别用冷冻保存的和新鲜的成纤维细胞作为核供体，所得重构胚卵裂率、囊胚率无显著差（P>0.05）；供体细胞离心800 r/min时所得重构胚效果较好，离心1500 r/min所得重构胚的卵裂率、囊胚率与其他3组相比较低；血清饥饿3～5 d组所得重构胚比其他3组好；用解冻复苏后再传2、5代的细胞进行核移植，所得重构胚的卵裂率、囊胚率无明显差异(P>0.05)，显著高于传8代的细胞所得重构胚。说明供体细胞的不同处理方法对核移植重构胚发育有很重要的影响。 相似文献
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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
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应用绵羊精子提取物体外激活绵羊体细胞核移植胚和山羊-绵羊异种核移植胚，比较了不同浓度的绵羊精子提取物对绵羊同种和异种重构胚的激活作用，并与传统的化学激活方法对重构胚的卵裂率、囊胚率和囊胚细胞数做了比较。结果表明，绵羊精子提取物可以有效地激活绵羊体细胞核移植胚和山羊-绵羊异种核移植胚，每一重构胚注射3pL浓度为5mg／mL的绵羊精子提取物，对同种和异种胚的发育效果均为最好，并显著高于其他试验组（P〈0．05）。注射绵羊精子提取物对绵羊-山羊异种核移植胚的卵裂率、囊胚率和囊胚细胞数的影响，与离子霉素联合6-DAMP激活法差异不显著（P〉0．05）。注射绵羊精子提取物对绵羊同种核移植胚的卵裂率和囊胚率的影响，与离子霉素联合6-DAMP激活法差异也不显著（P〉0．05），但精子提取物注射法的绵羊核移植胚的囊胚细胞数显著（P〈0．05）高于化学激活法的细胞数（72．4vs65．2）。 相似文献
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为提高绵羊体细胞核移植效率,以绵羊卵丘细胞为核供体,在融合后的核质互作期间加入咖啡因,在激活后对重构胚进行培养的过程中,加入组蛋白去乙酰化酶抑制剂Scriptaid。结果表明:咖啡因浓度以2.5 mmol/L或5 mmol/L为宜,尽管卵裂率、桑椹胚率以及囊胚发育率与对照组差异不显著(P>0.05),但囊胚细胞数显著高于对照组(P<0.05);以0.2μmol/L Scriptaid处理组囊胚发育率最高,达24.31%,显著高于对照组和0.8μmol/L Scriptaid处理组(P<0.05),但与0.4μmol/L Scriptaid处理组相比差异不显著(P>0.05);囊胚细胞总数以0.2μmol/L Scriptaid处理组最高,显著高于0.8μmol/L Scriptaid处理组(P<0.05),但是与对照组和0.4μmol/L Scriptaid处理组差异不显著(P>0.05)。以上结果表明,咖啡因处理对绵羊核移植重构胚的囊胚发育率无显著影响,但可以显著提高囊胚细胞总数;Scriptaid可以显著提高绵羊核移植重构胚的囊胚发育率。 相似文献
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H T Cheong Y Takahashi H Kanagawa 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》1992,54(6):1099-1103
This study was undertaken to examine the developmental capacity of reconstituted mouse embryos, and the influences of nucleus and cytoplasm on the development of these embryos following reciprocal pronuclear transplantation between in vitro 2-cell blocked and nonblocked embryos. Karyoplast containing pronuclei was transferred into the perivitelline space of the enucleated zygote and fused to cytoplasm with electrofusion. Maximum fusion rate was obtained when a field strength of 1.5 kV/cm was used. The fusion rates were high (86.2 +/- 3.2 to 90.6 +/- 2.0%) regardless of the strains of donor nucleus and recipient cytoplasm. Developmental rates of reconstituted embryos to the blastocyst stage, which were similar to that of the F1 (C57BL/6J x CBA) control were high when F1 embryos were used as the cytoplasm recipients (88.8 +/- 1.5 and 91.9 +/- 2.0%). When ICR embryos were used as the recipient cytoplasm, developmental rates were significantly reduced (71.5 +/- 2.9 and 54.1 +/- 3.2%), and affected by the source of nucleus. There were no significant differences in the cell number of embryos that developed to blastocysts and in the developmental rates to live young among the embryos reconstituted with different nuclei and cytoplasm, and the ICR control. The results of this study show that the development of reconstituted embryos is hardly affected by nuclear transplantation and electrofusion procedures. It is indicated that the recipient cytoplasm, rather than the donor nucleus, has the greater influence on the in vitro development of the reconstituted embryos to the blastocyst stage. 相似文献
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将发育不同时期的兔胚和移核胚的卵裂球细胞核与去核的成熟卵母细胞共同组成移核胚,通过中间受体培养和移植实验检验胚胎的发育能力。结果表明,（１）兔囊胚之前各个时期的胚胎细胞核均可使移核胚发育到囊胚;（２）胚胎极化前后的卵裂球参与组成的移核胚发育到囊胚的比例无显著差异。但极化后 ６４细胞胚的卵裂球与去核卵母细胞的融合率低于极化前的 ８细胞胚胎卵裂球;（３）兔 １６细胞胚与去核的卵母细胞组成的移核胚可以发育到期,产仔率为 ３．１６％ ;（４）兔移核胚卵裂球用于连续核移植,其后 ２ 代均可发育成囊胚,其中第 １ 代移核胚与第 ２ 代移核胚发育率相似,但显著高于第 ３ 代移核胚;（５）兔移核胚和各代连续移核胚卵裂球与去核卵的融合率无显著差异。 相似文献
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Parthenogenetic activation is an important factor in successful production of cloned mammals. Because it has been reported that aged oocytes are more sensitive to parthenogenetic activation than young oocytes, the present study examined the effects of oocyte aging on the in vitro and in vivo developmental potential of nuclear-transferred (NT) mouse oocytes receiving cumulus cells. The potentials of young NT oocytes (14 h after human chorionic gonadotrophin [hCG] injection) to develop into blastocysts was, however, significantly higher than that of aged oocytes (20 h after hCG injection; 16% vs 6%). When the nuclei of NT oocytes at the 2-cell stage were fused with enucleated fertilized 2-cell embryos, the potentials of the serial NT embryos to develop into blastocysts were no different for both young and aged oocytes (74% vs 74%). Live young, however, were obtained only after transfer of serial NT blastocysts developed from young NT oocytes (2%). In contrast to a report using embryonic nuclei as the nuclear donors, the results of the present study indicate that young oocytes are superior to aged oocytes as a source of recipient cytoplasm for mouse somatic cell cloning. 相似文献
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Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos 总被引：1，自引：0，他引：1
Atabay EC Katagiri S Nagano M Takahashi Y 《The Japanese journal of veterinary research》2003,50(4):185-194
Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes. 相似文献
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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
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An electrofusion methodology for transferring meiosis-II chromosomes (M-II-t) has not been completely established. The present study compared the use of two temperatures (fusion at 37 C for Group A and 25 C for Group B) during an electrofusion procedure for mouse oocyte M-II-t and investigated the cytogenetic normality and developmental competence of embryos derived from in vitro fertilization using oocytes reconstructed by M-II-t. The M-II-t oocytes were fertilized in vitro and cultured to the blastocyst stage; the resultant embryos were analyzed cytogenetically. Subsequently, chromosomal normality of the resultant embryos at the prometaphase stage of first cleavage division and the integrity of the meiosis-II spindles of the reconstructed oocytes were analyzed. The success rate of electrofusion in Group B was 92.1%, which was significantly different from that in Group A (49.2%) (P<0.05). The fertilization rates (A, 80.7%; B, 77.2%) and development rates (A, 70.9%; B, 65.5%) in the M-II-t groups were significantly lower than those in the control group (95.0 and 92.2%, respectively) (P<0.05). The incidence of chromosomal abnormalities in the Group A embryos (20.5%) at the blastocyst stage was significantly higher than that in the control group embryos (8.5%) (P<0.05), but the incidence of chromosomal abnormalities in Group B (12.5%) was not significantly different compared with the other groups. A temperature of 25 C during the electrofusion procedure for M-II-t resulted in a good fusion rate, good development rate, and efficient production of chromosomally normal blastocysts. Furthermore, the incidence of chromosomal abnormalities in the first cleavage embryos at the prometaphase stage in Group B (9.6%) did not differ significantly from that in the control group (6.6%). The spindle morphology of the M-II-t oocytes in Group B was normal. 相似文献
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In Vitro Development of Marbled Cat Embryos Derived from Interspecies Somatic Cell Nuclear Transfer 总被引：1，自引：0，他引：1
A Thongphakdee P Numchaisrika S Omsongkram K Chatdarong S Kamolnorranath S Dumnui M Techakumphu 《Reproduction in domestic animals》2006,41(3):219-226
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
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猪卵丘细胞核移植研究 总被引：1，自引：0，他引：1
以卵丘细胞为供核体细胞,采用胞质内注射法进行猪体细胞核移植,对去核、激活和培养等关键技术过程进行研究,结果表明,①点压法、挤压法对卵母细胞去核率明显高于盲吸法(三者分别为62.5%,64.6%,50.7%,P<0.05)。盲吸法去核染色体容易发生位置偏离,影响去核效果,但对早成熟的卵母细胞(36h～44h)进行去核可明显提高去核效率(P<0.05),在成熟培养36h～38h、39h～41h、42h～44h去核率分别为60.9%、67.8%、64.3%,而45h～48h为48.4%。②体细胞预激活有助于提高核移胚卵裂率(28.0%,20.1%,P<0.05)。A23187(Ca2 ionophore,钙离子载体)单独或与6DMAP(6Dimethylaminopurine,6二甲基氨基嘌呤)联合作用能使猪体细胞核移胚激活继续发育。③核移胚以胚胎培养液NCSU23(NorthCarolinaStateUniversity23medium,北卡洲立大学培养液23)及卵丘颗粒单层共培养体系进行分别培养,核移胚卵裂率无明显差异(30.06%,31.5%,P>0.05)。但NCSU23培养4细胞后发育能力更高(13.5%,3.9%,P<0.05)。 相似文献
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供体细胞培养处理方法对水牛核移植效果的影响 总被引：4，自引：1，他引：4
以经常规培养法 (DMEM 10 % FCS)、血清饥饿法 (DMEM 0 .5 % FCS培养 5～ 10 d)和 Apidicolin- APD结合血清饥饿法 (0 .1mg/ L APD培养 2 4 h,DMEM 0 .5 % FCS培养 1～ 18d)培养处理的水牛卵巢颗粒细胞和水牛成体耳部成纤维细胞作供核 ,分别采取带下注核法和胞质内注核法进行核移植。同一供核细胞各处理组间的核移植胚融合率 (以颗粒细胞作供核 )以及重组胚的囊胚发育率无明显差异 (P>0 .0 5 ) ,但经 APD 0 .5 % FCS培养处理供体细胞核移植后的分裂率显著高于其他组 (P<0 .0 5 )。用 7%乙醇处理的成体耳部成纤维细胞进行核移植 ,其重组胚的分裂率和囊胚发育率与对照组 (不含乙醇 )均无明显差异 (P>0 .0 5 )。结果表明 ,(1)血清饥饿处理水牛供体细胞对其核移植效果没有影响 ;(2 ) DNA合成抑制剂 APD结合血清饥饿培养处理水牛颗粒细胞和成体耳部成纤维细胞 ,可提高其核移植效果 ;(3)乙醇预激活处理水牛成体耳部成纤维细胞 ,对其核移植效果没有影响 相似文献
20.
This study was conducted to reconstruct heterogeneous embryos using equine skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplast for investigating the reprogramming of equine somatic cell nuclear in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Adult horse skin fibroblast cells serum-starved were used as donor somatic cells. Bovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electofusion. The fused eggs were activated by inomycin with 2 mm/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The results showed that the first completed cleavage of xenonuclear transfer equine embryos occurred between 30 and 48 h following activation. 52% of the injected oocytes were successfully fused, 72% of the fused eggs underwent the first egg cleavage and 17% of the heterospecific nuclear-transferred zygotes developed to 4- or 8-cell embryo stages. This study demonstrated that the reconstructed embryos have undergone the first embryonic division and the reprogramming of equine fibroblast nuclei can be initiated in bovine-enucleated oocytes. 相似文献