电激活对完全体外化牛细胞核移植的影响

牛卵母细胞在体外成熟２３～２４ｈ时去核，去核卵用８０Ｖ／ｍｍ４０μｓ两次电脉冲激活（Ⅰ组）或不激活（Ⅱ组），然后将体外受精、发育的８～３２细胞期胚胎的单个卵裂球注入卵周隙，用８０Ｖ／ｍｍ４０μｓ两次电脉冲诱导卵裂球与去核卵母细胞融合。Ⅰ组操作后存活率和融合率（８８．４％和５５．０％）显著低于Ⅱ组（９５．９％和６５．１％，Ｐ＜０．０５）。融合卵体外培养２４ｈ和５～８ｄ后，Ⅰ组核移植胚胎的卵裂率和桑椹／囊胚发育率（６３．０％和１５．２％）与Ⅱ组（５０．５％和７．８％）无显著差异，但Ⅰ组结果均好于Ⅱ组。将来自两组的２５枚桑椹和囊胚移植到１６头同期受体，在已检查过的８头受体中２头受体妊娠，其中１头于妊娠后４个多月流产，１头于妊娠期满后产出一雄性牛犊。研究结果表明：去核卵母细胞的电激活尽管对重组卵的存活与融合不利，但可改善核移植胚的卵裂和发育。本研究在我国首次获得牛细胞核移植的成功，证明用ＩＶＭ卵母细胞和ＩＶＦ胚胎进行完全体外化的牛细胞核移植是可行的。     

兔核移植胚胎的克隆研究

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。     

牛体外受精胚胎细胞核移植的实验研究

将采自肉联厂屠宰车间的牛卵巢中的卵母细胞置于ＴＣＭ－１９９＋１０％ＥＣＳ（０ｄ）＋ＦＳＨ（１万ＩＵ／Ｌ）＋ＬＨ（５ｍｇ／Ｌ）中培养成熟，用含０．３％透明质酸酶的消化液将卵母细胞周围的卵丘细胞去掉，并以７．５ｍｇ／Ｌ的ＣＢ液处理后，用微吸管吸去透明带内的第一极体及极体下面的部分卵母细胞质，然后将经体外受精并发育至８～１６细胞期胚胎的卵裂球注入去核卵母细胞的卵周隙中，再将移核胚置于电融合槽内进行融合（电场强度为１２００Ｖ／ｃｍ，持续时间为８０μｓ，１次脉冲刺激）。将融合的胚胎移入生长有单层贴壁颗粒细胞的发育培养液（ＴＣＭ－１９９＋１０％牛血清）中培养，观察移核胚的融合率及发育率，部分去核卵母细胞经染色后观察去核率。结果表明：（１）移核胚的融合率为８６．９％（５３／６１）；（２）发育至２～８细胞期的胚胎占培养的移核胚胎总数的２１．３％（１０／４７）；（３）卵母细胞的去核率为６８．２％（４５／６６）。     

家兔胚胎细胞核移植与连续移植

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。     

核移植兔研究进展

哺乳动物胚胎细胞核移植(NuclearTransplantation,NT)是指将动物早期胚胎卵裂球以显微手术和细胞融合的方法移植入去核的卵母细胞,并使其能正常分裂、发育成为新个体的技术。核移植技术对于研究动物繁殖和胚胎在发育过程中的核质互作有重要意义。尽管核移植在多种动物都已...     

影响核移植后小鼠重构胚存活因素的研究

将不同类型的供体细胞(卵丘细胞、心肌细胞和上皮细胞)的遗传物质注射到去核的MⅡ卵母细胞中,获得各种重构胚胎,以分析对小鼠卵母细胞进行核移植操作获得重构胚过程中的各种影响因素。结果表明,用Gi emas染料将挤出的卵母细胞质染色证实,可以完整地去除MⅡ卵母细胞核。对370个MⅡ卵母细胞进行去核,去核成功率为92.4%,注核成功率为82.1%;给予90 v/mm,80μsec,1次电脉冲刺激,融合率为72.1%;将融合激活后的重组胚转入KSOM培养基中,平均囊胚率达到35.8%。在胚胎重构过程中,出现了核仁显著变大,出现不同数量核仁的现象。通过比较各种重构胚胎体外后续发育,结果卵丘细胞重构胚胎的体外发育率最好。     

昆明小鼠内细胞团和滋胚层细胞注入去核2-细胞胚的核移植研究

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。     

核移植前激活受体卵子对牛核移植卵体外发育的影响

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     

小鼠-猪种间体细胞克隆试验

用3代~5代小鼠胎儿成纤维细胞(MEF)作核供体细胞,体外成熟培养44 h~46 h、排出第1极体(PbI)的猪卵母细胞去核后,进行小鼠-猪种间核移植操作。Hoechst 33342染色检查去核效果,去核率为84.2%(27/32)。共培养68枚小鼠-猪种间核移植重构胚,其中48枚发生卵裂,卵裂率为70.6%,13枚胚胎发育至8-细胞,发育率达27.1%(13/48)。     

猪胚胎核移植研究进展

核移植就是将共体核移入受体卵胞质获得重组胚的过程，核移植可以产生遗传组成相同的若干后代，前景迷人。猪胚胎核移植目前以早期卵裂球做供体，次级卵母细胞做受体。猪胚母体－合子转变（ＭＺＴ）发生于４细胞期，ＭＺＴ不影响重组胚的发育能力。核质融合及激活老头儿采电激法，可有效地使重级胚发育，卵母细胞质可使融入的核发生再规范产重排发育程序，供体细胞周期对核移胚的发育有重要影响。由于猪胚的特殊性，核移植的难度较大     

Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo Production In Vitro

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNβ-Z or LNβ-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro . Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNβ-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 ± 6.4%; 12.0 ± 5.7%) or EGFP (57.5 ± 6.3%; 10.1 ± 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 ± 8.2%; 12.3 ± 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.     

RNA Synthesis in Porcine Blastomere Nuclei Introduced into in Vitro Matured Ooplasm

The objective was to investigate the RNA synthesis in porcine blastomere nuclei upon transplantation into in vitro matured enucleated oocytes. Nuclei from 2- to 8-cell porcine embryos were introduced into the ooplasm of in vitro matured and enucleated porcine oocytes by electrofusion, and the resultant reconstructed embryos were cultured in vitro. Before fusion or at different intervals after this event embryos were incubated with [³H]-uridine, fixed, and histologically processed for autoradiography in order to detect RNA synthesis. About two thirds of the embryos were considered to depict normal development. All blastomeres displayed pronounced RNA synthesis before fusion, at 3 and 9 h after fusion the synthesis decreased or ceased, and at 24-49 h some embryos resumed synthesis at the 1- to 2-cell stage.     

Heterospecific Nuclear-transferred Embryos Derived from Equine Fibroblast Cells and Enucleated Bovine Oocytes

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.     

Dynamic analysis of Ca2+ level during bovine oocytes maturation and early embryonic development

Mammalian oocyte maturation and early embryo development processes are Ca²⁺-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca²⁺ was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca²⁺ was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca²⁺ was present throughout the blastomere. In PA embryos, Ca²⁺ was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca²⁺ in the SCNT embryos. However, Ca²⁺ was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca²⁺ showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca²⁺ location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.     

Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes

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.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

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.     

Development and Status of Cattle Embryo Cloning in Germany

Contents: A review about experiments in bovine embryo cloning performed by different working groups in Germany is given. The procedure is shortly described and the achieved results are specified. Average enucleation rates in the experiments were 58–74%, electrofusion rates were 31 to 85%. Between 3 and 17% of the in vitro cultured embryos cleaved to transferable embryos. The first calf emerging from nuclear transfer in Germany was born in August, 1992. A clone of three identical calves was given birth in April, 1993. Four months later a calf was born, which exclusively emerged from in vitro techniques (in vitro maturation of recipient oocytes, in vitro production of blastomere donor embryo, in vitro culture of cloned embryos). Finally some future aspects of bovine embryo cloning in Germany are illustrated.     

秋水仙胺对电融合方法体外制备牛四倍体胚胎的影响

通过添加秋水仙胺从而改善电融合方法体外制备牛四倍体胚胎的过程。首先检测牛早期胚胎的分裂时间,并由此确定于体外受精后26~30h时间段内挑选既同期化且优质的2细胞期胚胎用于电融合。电融合参数选取2次直流电压为0.75kV/Cm且脉冲时间60μs。并通过免疫荧光染色监测电融合后细胞核的重组过程。设立电融合对照组和电融合秋水仙胺处理组,处理组在电融合后随即处理0.05mg/L秋水仙胺6h,后彻底洗涤继续体外培养至囊胚期。处理组的融合率（84.4%vs 84.8%）、分裂率（74.3%vs 77.6%）和囊胚率（36.1%vs 39.4%）皆低于对照组,但差异均不显著。获取的囊胚期胚胎的核型分析结果显示,处理组的四倍体制备率显著高于对照组（59.4%vs 26.9%）。综上所述,对电融合后的胚胎处理0.05mg/L秋水仙胺6h显著提高了牛四倍体电融合方法体外制的备率。     

Effect of transfection and passage number of ear fibroblasts on in vitro development of bovine transgenic nuclear transfer embryos

The objective of this study was to determine if the transfection of human prourokinase (ProU) gene and passage number of transfected ear fibroblasts affected in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human ProU was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker and human ProU gene into a pcDNA3 plasmid and transfected into bovine ear fibroblasts using a lipid mediated method. Abattoir derived oocytes were enucleated at 18-20 hr post maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the couplets were cultured in modified synthetic oviductal fluid (mSOF) medium for 168 hr. In Experiment 1, significantly lower rate in blastocysts formation (10.3%) was observed in transfected donor cells at early passage than that in nontransfected counterparts (22.1%, P<0.05). In Experiment 2, development to blastocysts and GFP expression in blastocysts were not significantly different between early (3-7) and late (8-12) passage donor cells (10.3 vs. 11.3% and 54.5 vs. 41.7%, respectively). This study indicates that in vitro development of bovine transgenic NT embryos is negatively influenced by transfection of human ProU gene into donor fibroblasts. However, passage number of transfected ear fibroblasts does not affect in vitro development of bovine transgenic NT embryos.     

Geklontes Kalb in Neustadt geboren

Am 27.8.1992 wurde beim Besamungsverein Neustadt (BVN) in Neustadt a.d. Aisch ein geklontes Fleckviehkalb geboren. Die Identität der Abstammung konnte eindeutig gesichert werden. Der erfolgreiche Kerntransfer in einem Versuchsprogramm soll belegen, daβ sich nach der Fusion einer Blastomere aus einem Spenderembryo mit einer in vitro gereiften und enukleierten Empfängeroozyte ein Embryo entwickeln kann, der nach Transfer auf ein Empfängertier zur Geburt eines gesunden Kalbes führt.
Contents: A cloned calf was born in Neustadt ad. Aisch
A cloned "Fleckvieh" calf was born at the Al-Association Neustadt (BVN) in Neustadt a.d. Aisch on August, 27, 1992. The identity of the parentage could be verified undoubtedly. The successful nuclear transfer in an experimental program showed that the fusion of a blastomere from a donor embryo with an in vitro matured and enucleated recipient oocyte led to the development of an embryo, and after transfer to a synchronous recipient to the birth of a healthy calf.