Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.     

In Vitro Development of Bison Embryos Using Interspecies Somatic Cell Nuclear Transfer

Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non‐domestic animal species. However, problems arise during the development of these embryos, which may be related to species‐specific differences in nuclear–cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria‐related genes NRF1, MT‐CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34–33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45–12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80–87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT‐CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.     

卵母细胞核因子促进核移植四倍体胚胎早期发育研究

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。     

Study on Oocyte Nuclear Factor Promotes the Early Development of Tetraploid Somatic Cell Nuclear Transfer Embryos

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.     

体细胞核移植技术生产猪植酸酶转基因胚胎的研究

为获得具有植酸酶腮腺特异性表达的猪转基因克隆胚胎,本研究使用植酸酶腮腺特异性表达的DNA质粒(包含腮腺分泌蛋白(parotid secretary protein,PSP)启动子与终止子序列、Neo筛选基因、绿色荧光蛋白(EGFP)报告基因和高比活的植酸酶appA基因),采用脂质体转染和基因素418(G418)药物抗性筛选的方法获取稳转细胞系,并利用体细胞核移植技术获得植酸酶转基因胚胎。结果表明,本研究构建的DNA质粒可用于细胞筛选,且质粒越小,细胞的转染效率越高,14.89 kb的YM6552仅获得了7.1%的转染率,EGFP质粒则获得了43.4%的转染效率。在单克隆形成上,较小的pYN3600也获得了更高的单克隆形成数(25个),其中表达EGFP的单克隆有14个,植酸酶PCR阳性集落有11个,高于YM6552的单克隆数(19、8和6)。转基因细胞构建重构胚胎后,所有的胚胎均能表达绿色荧光蛋白,虽其体外发育能力有所下降,但差异不显著(P>0.05)。综上所述,本研究所采用的植酸酶质粒、细胞筛选方法和核移植技术可生产植酸酶重构胚。     

卵母细胞来源对水牛体细胞核移植胚胎发育潜能的影响

【目的】探索不同来源卵母细胞对体细胞核移植(SCNT)重构胚的发育能力及发育潜能关键蛋白表达水平的影响。【方法】试验分为活体采卵(OPU)和屠宰场(SLH)卵巢2组,OPU组用超声波活体采卵仪穿刺抽吸10头非泌乳期经产水牛卵巢的卵泡采卵,SLH组从屠宰场卵巢抽吸卵泡采卵。获得的卵母细胞分别进行体外成熟,体外成熟22～24 h后,吹打去除卵丘细胞,挑选具有第一极体的卵母细胞,去核后与水牛耳部成纤维细胞进行SCNT,分别统计SCNT重构胚的融合率、分裂率和囊胚率,用免疫荧光检测2种SCNT重构胚的E-钙黏蛋白(E-cadherin)和转录因子Sox2蛋白的表达水平。【结果】OPU组卵母细胞成熟率及其SCNT重构胚的囊胚率均显著高于SLH组(P<0.05),但2组SCNT重构胚的融合率和分裂率均无显著差异(P>0.05);免疫荧光结果显示,E-cadherin蛋白定位于细胞膜上,Sox2蛋白分布在细胞核膜及细胞质中,OPU组SCNT重构胚中E-cadherin和Sox2的表达水平均显著高于SLH组(P<0.05)。【结论】活体采集的水牛卵母细胞更适合用于SCNT重构胚的构建...     

绵羊卵丘细胞细胞周期同步化对核移植胚胎发育的影响

供体细胞周期同步化是影响体细胞核移植成功率的重要因素之一.试验分别对绵羊卵丘细胞采用血清饥饿和接触抑制的方法进行细胞周期同步化处理,使用流式细胞仪检测各组细胞周期的分布.结果发现,与对照组相比,卵丘细胞经血清饥饿24~72 h后,显著地增加了G₀/G₁期细胞的百分比(P <0.05);接触抑制24~72 h,G₀/G₁期细胞所占比例与血清饥饿组无显著差异(P >0.05),但显著高于对照组(P <0.05);用经血清饥饿与接触抑制的供体细胞进行核移植后,重构胚卵裂率、桑椹胚率和囊胚率差异不显著(P >0.05),但二者囊胚率显著高于对照组(P <0.05).上述结果证实,血清饥饿和接触抑制均能使绵羊卵丘细胞周期同步化至G₀/G₁,均可用作绵羊体细胞核移植的供体细胞细胞周期同步化处理.     

In Vitro Development of Marbled Cat Embryos Derived from Interspecies Somatic Cell Nuclear Transfer

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.     

An Inter‐Subspecies Cloned Buffalo (Bubalus bubalis) Obtained by Transferring of Cryopreserved Embryos via Somatic Cell Nuclear Transfer

The aim of this study was to explore the feasibility of cryopreservation of inter‐subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum‐starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross‐bred buffalo (Murrah × Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13‐month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter‐subspecies cloned embryos is feasible to produce buffalo offspring.     

自由基吸收类抗氧化剂对延边黄牛体细胞克隆重构胚发育的影响

为提高延边黄牛体细胞克隆重构胚发育率,研究了在体外培养液中单独添加不同浓度的自由基吸收类抗氧化剂维生素E(VE)、表没食子儿茶素没食子酸酯(EGCG)和亚硒酸钠(SS)对克隆重构胚发育率的影响。试验中选择成熟培养20～22h的卵母细胞,脱颗粒细胞后选择排出第一极体的卵母细胞进行去核与注核操作,然后将融合、激活后的重构胚在添加不同种类不同浓度抗氧化剂的体外培养液中进行培养。结果表明,在卵裂率上,VE的100μmol.L-1组显著高于其他3组(84.58%vs 57.64%、70.87%、67.64%);50和200μmol.L-1组之间无差异,但都显著高于0μmol.L-1组(70.87%、67.64%vs 57.64%)。EGCG的7.5和15μmol.L-1组显著高于加0μmol.L-1组(79.47%、81.67%vs 69.47%)且其他2组间差异不显著。SS的添加,5ng.mL-1组与0和10ng.mL-1组有显著差异(80.44%vs 70.27%、67.16%);与2.5ng.mL-1组差异不显著(80.44%vs 73.92%),且其他2组间差异不显著;在囊胚发育率上,VE的100μmol.L-1组要显著高于其他3组(26.36%vs 13.04%、18.08%、17.26%)。EGCG的15μmol.L-1组显著高于0和30μmol.L-1组(24.86%vs 8.99%、12.10%),和7.5μmol.L-1组无差异。SS的5与0组和10ng.mL-1组有显著差异(23.31%vs 10.32%、12.65%);2.5ng.mL-1组与0和10ng.mL-1l组有显著差异(19.97%vs 10.32%、12.65%)且其他2组间差异不显著。体外培养液中分别单独添加浓度为100μmol.L-1、15μmol.L-1和5ng.mL-1的维生素E,EGCG和亚硒酸钠都能够显著提高延边黄体细胞克隆牛重构胚的发育率。     

Chronological Appearance of Apoptosis in Bovine Embryos Reconstructed by Somatic Cell Nuclear Transfer from Quiescent Granulosa Cells

Efficiency of cloning has remained low and in spite of attempts to improve this technology, many reconstructed embryos do not implant or are lost during early pregnancy. Chromosomal aberrations, deviant gene expression patterns and abnormal regulation of cell death may be involved in this increased early embryonic loss. Here, we investigate the chronological onset of both apoptotic changes in nuclear morphology and DNA degradation [detected by transferase-mediated dUTP nick-end labelling (TUNEL) reaction] in bovine two-cell- to blastocyst-stage embryos. Such embryos were generated either by reconstruction with nuclear transfer from quiescent granulosa cells or by regular in vitro embryo production. Nuclear condensation was observed from the two-cell stage and TUNEL labelling was observed from the six-cell stage in reconstructed embryos, whereas nuclear condensation was evident from the eight-cell stage and TUNEL labelling from the 13-cell stage in embryos derived in vitro. Furthermore, reconstructed embryos displayed elevated ratios of embryos containing apoptotic nuclei at pre-compaction stages and higher indices of apoptotic nuclei in morula and blastocyst stages when compared with in vitro-produced embryos.     

A Cathepsin B Inhibitor,E-64, Improves the Preimplantation Development of Bovine Somatic Cell Nuclear Transfer Embryos

Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.     

提高猪核移植效率的研究进展

哺乳动物体细胞核移植(克隆)经过多年的发展,获得了多种后代。猪的核移植研究意义重大,应用价值颇高,是当今生物技术的热点研究之一。尽管猪核移植已取得了很大进展,但移植效率依然很低。本文就目前已有的资料,综述了提高猪体细胞核移植效率的各项技术环节的研究进展。     

BCB和Zebularine对绵羊体细胞核移植胚胎发育效果的影响

为探究亮甲酚蓝(BCB)染色选择的卵母细胞是否有利于体细胞核移植胚胎的体外发育,本实验将卵丘-卵母细胞复合体(COCs)放入含有BCB的PBS中染色,根据细胞质颜色可将卵母细胞分成BCB~+组和BCB~-组,并以未经BCB处理的COCs作为对照组,然后将卵母细胞进行体外成熟,统计卵母细胞的成熟率。将成熟后的卵母细胞进行体细胞核移植,其中,部分BCB~+组卵母细胞所需的供体细胞利用Zebularine处理,统计体细胞核移植的卵裂率、桑椹胚率和囊胚率。结果表明:BCB~+组卵母细胞的成熟率显著高于对照组和BCB~-组(71.15%vs 65.38%,53.52%,P0.05);BCB~+组核移植胚胎的卵裂率(87.91%vs 56.83%)、桑椹胚率(37.41%vs 21.73%)和囊胚率(21.48%vs6.82%)均显著高于BCB~-组(P0.05);与对照组相比,BCB~+组卵裂率(87.91%vs 83.23%)和囊胚率(21.48%vs14.89%)也显著升高(P0.05)。BCB~+组供体细胞经Zebularine处理后,胚胎发育能力进一步提高,其中囊胚率显著高于BCB~+组、对照组和BCB~-组(29.25%vs 21.48%,14.89%,6.82%,P0.05)。     

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

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。     

Nuclear Donor Cell Lines Considerably Influence Cloning Efficiency and the Incidence of Large Offspring Syndrome in Bovine Somatic Cell Nuclear Transfer

Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post‐natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre‐implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p < 0.05). The pregnancy rates at 90 and 240 day were significantly lower in groups F2 (4.9% and 3.3%) and F3 (5.4% and 5.4%) compared to groups F1 (23.3% and 16.3%), F4 (25.7% and 18.6%) and F5 (25.9% and 19.8%) (p < 0.05). The cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p < 0.05). Moreover, large offspring syndrome (LOS) incidence in group F5 was significantly lower than those in other groups (p < 0.05). All cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves.     

Fine Structures of Embryonic Discs of In Vivo Post‐hatching Porcine Blastocysts at the Pre‐Primitive Streak Stage

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre‐primitive streak area of embryonic discs. The cells were 1–2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc.     

Production of Wild Buffalo (Bubalus arnee) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Domestic Buffalo (Bubalus bubalis) Oocytes

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin‐18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open‐pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re‐expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified–warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.     

水牛体细胞核移植方法比较及激活前的时间间隔对全细胞胞质内注射法核移植效果的影响

本研究旨在比较水牛2种体细胞核移植(Somatic Cell Nuclear Transfer,SCNT)方法的效果以及激活前的时间间隔对全细胞胞质内注射法(Whole-Cell Intracytoplasmic Microinjection,WCICSI)核移植效果的影响.采用水牛胎儿成纤维细胞作为供核,比较了透明带下注核法(Perivitelline Microinjection,PM)和WCICSI核移植效果.另外,试验了不同类型的供体细胞进行全细胞胞质内注射后与激活前的受体胞质的最适宜作用时间.结果,WCICSI构建核移植重构胚的成功率显著高于PM(87.1%vs 81.1%,P＜0.05),虽然其重构胚的分裂率极显著低于PM(49.5%vs 71.8%,P＜0.01),但囊胚率、核移植的效率无显著差异(P＞0.05).卵丘细胞和胎儿成纤维细胞在注射后3 h激活,重构胚的囊胚发育率最高;颗粒细胞注射后与激活前的最佳时间间隔可在1.5～3 h,但3 h是最佳的作用时间.结果表明,(1)WCICSI可用于水牛体细胞核移植的研究;(2)水牛胞质内注射供体细胞后3 h进行激活,核移植重构胚的发育效果最好.