胚胎干细胞与四倍体互补产生转基因小鼠

目的 将四倍体胚胎补偿技术与转基因相结合，构建基因打靶载体转化的胚胎干细胞(ESCs)，获得小概率的同源重组事件，进而直接得到所要的突变品系。 方法 通过电穿孔的方法将增强型绿色荧光蛋白（EGFP）表达质粒转入ESCs，用G418筛选获得EGFP-ESCs。另外，通过电融合获得四倍体囊胚细胞，显微注射法将19～21个EGFP-ESCs注入每个四倍体囊胚腔，分别移植到假孕2.5d雌鼠子宫或假孕0.5d的输卵管内。 结果 获得稳定表达的EGFP-ESCs，染色体数目正常 (2n＝40条)；四倍体细胞融合率为95.07%，囊胚发育率为95%；共获得410个重构囊胚；移植后未得到出生小鼠，但共观察到了151个着床位点，子宫移植的着床率为29.41%，输卵管移植的着床率为64.37%，获得的胚胎中EGFP蛋白有散在的表达。 结论 稳定表达的EGFP-ESCs参与到了转基因胎鼠的发育中；本实验中输卵管移植的着床率高于子宫移植的着床率。     

昆明小鼠3.5 d和4 d囊胚不同分离方法的比较

背景：远交系的昆明小鼠作为中国自然科学研究主要实验动物,其胚胎干细胞建系的成功率一直很低。 目的：探讨昆明小鼠胚胎干细胞体外分离培养的最佳方法和最适合的采胚时间。 方法：采集孕3.5 d和4 d的囊胚分别以免疫外科法和全胚培养法在小鼠胚胎成纤维细胞饲养层上分离和克隆昆明小鼠胚胎干细胞集落。 结果与结论：免疫外科法和全胚法培养3.5 d囊胚的内细胞团贴壁率和原代克隆形成率差异均不显著(P  > 0.05);全胚法培养4 d囊胚的内细胞团贴壁率显著高于免疫外科法(P < 0.05),但原代克隆形成率显著低于免疫外科法(P  < 0.05);全胚法培养4 d囊胚的原代克隆形成率显著高于3.5 d囊胚(P  < 0.05);免疫外科法分离4 d囊胚内细胞团的贴壁率和原代克隆形成率显著高于以同样方法分离培养的3.5 d囊胚(P  < 0.05)。结果显示用免疫外科法分离4 d囊胚更适合于昆明小鼠胚胎干细胞的体外分离培养。     

小鼠胚胎干细胞饲养层培养体系的优化筛选

目的：建立小鼠胚胎干细胞饲养层培养体系。方法：用5种不同鼠胚成纤维细胞为饲养层，进行小鼠胚胎干细胞的分离培养，观察5种饲养层培养体系对小鼠胚泡发育，内细胞团增殖及胚胎干细胞分离培养的作用。结果：原代或冻存复苏后的原代培养鼠胚成纤维细胞用于制备饲养层，有利于胚泡的贴壁，孵化，内细胞团增殖形成巢式生长集落，离散后培养，可以观察到胚胎干细胞集落的出现，并可在短期内维持胚胎干细胞的正常形态，不发生分化，与其他三组有明显差异。结论：原代或冻存复苏后的原代培养鼠胚成纤维细胞饲养层是用于胚胎干细胞分离培养的有效的培养体系。     

卵裂期胚胎解冻后囊胚培养临床结局的相关因素的研究

目的探讨研究解冻后卵裂期胚胎行囊胚培养后临床结局的相关因素分析。方法收集我院生殖医学中心2015年3月-2017年5月解冻并进行囊胚培养的分裂期胚胎,如继续发育后行胚胎移植,并随访其临床结局。结果临床妊娠和出生率与是否移植完整胚胎、是否移植优质囊胚无明显相关(P0.05)。至少移植一枚以上囊胚组的临床妊娠率为58.4%,出生率为49.6%;8例移植胚均未发育至囊胚组无临床妊娠(P0.01,fisher′s exact test)。未形成囊胚组冷冻时和移植时年龄均显著高于囊胚形成组(P0.01);囊胚形成组的平均完整存活数明显高于未形成囊胚组(P0.05)。结论冷冻胚胎解冻后,囊胚的形成与解冻后胚胎完整度以及年龄相关。解冻后培养至5-6天的胚胎如无囊腔形成,则可能其种植潜能差,仍需加大样本量进一步研究。     

人胚胎成纤维细胞对人胚胎干细胞生长的作用

目的：比较人和小鼠胚胎成纤维细胞对人胚胎干细胞生长的作用,为胚胎干细胞定向诱导各系统细胞应用于临床,消除异种蛋白污染打下基础。方法：分别采用人胚胎成纤维细胞和小鼠胚胎成纤维细胞为饲养层细胞,支持人受精卵的培养,观察其增殖和分化情况。结果：人和小鼠胚胎成纤维细胞分别加入白血病抑制因子（hLIF）均能很好支持人胚胎干细胞生长增殖,并保持未分化状态。结论：完全可以使用人胚胎成纤维细胞支持人胚胎干细胞增殖,消除异种蛋白污染的可能性,为胚胎干细胞定向诱导分化发育应用于临床打下坚实基础。     

单卵母细胞对应的卵丘颗粒细胞中生长分化因子-9和骨形成蛋白-15的表达水平与胚胎发育潜能的研究

目的评估单卵母细胞对应的卵丘颗粒细胞中GDF9和BMP15的m RNA表达水平对卵母细胞发育潜能的影响。方法采用长方案治疗25例ICSI助孕的女性,取卵当天收集180份单卵母细胞对应的卵丘颗粒细胞,随后提取卵丘颗粒细胞的m RNA,进行实时荧光定量分析,对GDF9和BMP15 m RNA基因表达水平采用独立样本t检验分析。结果常规长方案CCs 180份,因卵子与收集的卵丘颗粒细胞是一一对应的,根据卵子受精情况将CCs分为正常受精组CCs 129份,异常受精组CCs 51份;正常受精组根据第3日胚胎卵裂情况分为优质卵裂胚形成组CCs 58份,非优质卵裂胚形成组CCs71份;非优质卵裂胚继续培养,根据第5日形成囊胚情况,分为优质囊胚形成组CCs 25份,非优质囊胚形成组CCs 46份。GDF9和BMP15 m RNA基因表达水平与正常受精率,优质胚胎率显著相关。正常受精组GDF9和BMP15 m RNA基因表达水平显著高于非正常受精组(P0.01),优质胚胎组显著高于非优质胚胎组(P0.01)。结论单卵母细胞对应的卵丘颗粒细胞中GDF9和BMP15 m RNA基因表达水平与正常受精率,优质胚胎率和优质囊胚形成率呈显著正相关,提示其可以作为预测卵母细胞的发育潜能的指标。     

小鼠Lewis肺癌细胞与胚胎干细胞体外共培养的生物学行为

背景:从发生学角度来看,肿瘤细胞和胚胎干细胞都受原癌基因调控,并具有惊人的分裂能力。将胚胎癌细胞移植入正常发育的同系小鼠的胚泡内,结果产生了不具有恶性表型的嵌合体小鼠,表明在胚胎环境中肿瘤细胞的命运可以发生改变。目的:探讨小鼠胚胎干细胞对Lewis肺癌细胞生物学行为变化的影响。方法:通过Transwell小室体外共培养小鼠胚胎干细胞与Lewis肺癌细胞。实验组Transwell小室中为成纤维细胞和胚胎干细胞,12孔板中为肿瘤细胞;对照组将加入到Transwell小室中的胚胎干细胞替换为成纤维细胞,保持小室中细胞的总数不变,12孔板中为肿瘤细胞。结果与结论:与对照组比较,实验组Lewis肺癌细胞形态变化显著,部分细胞出现老化甚至凋亡迹象;实验组Lewis肺癌细胞的增殖明显减慢(P0.05),穿透DB胶生物膜的Lewis肺癌细胞数明显减少(P0.05)。提示胚胎干细胞能够抑制Lewis肺癌细胞的生长增殖及侵袭力。     

小鼠饲养层细胞制备与小鼠胚胎干细胞SF1-G的培养

背景:小鼠胚胎干细胞系SF1-G是由雌性C57BL/6小鼠与雄性M.spretus小鼠交配后,取桑葚胚期胚胎在STO饲养层细胞上分离培养获得,STO细胞较昂贵,而由小鼠胚胎成纤维细胞制备的饲养层细胞不仅取材容易,而且形成胚胎干胞的克隆率、维持胚胎干细胞正常核型的能力均比STO细胞要好一些,因此,建立一种适宜SF1-G细胞扩增的培养体系,保持其未分化状态生长是充分利用胚胎干细胞资源的前提。目的:建立有效的小鼠胚胎成纤维细胞的分离培养及胚胎干细胞饲养层细胞制备体系;以建立有效的小鼠胚胎干细胞(SF1-G细胞)扩增培养体系。方法:从孕12.5～14.5d的ICR小鼠分离培养原代小鼠胚胎成纤维细胞;取3～5代的小鼠胚胎成纤维细胞,以丝裂霉素C抑制其增殖能力制备饲养层细胞;在饲养层细胞上增殖培养SF1-G细胞;染色体G显带分析法检测SF1-G细胞核型,SF1-G细胞碱性磷酸酶染色和RT-PCR检测Oct4、Nanog基因表达。结果与结论:从孕鼠胚胎有效分离到小鼠胚胎成纤维细胞,以3～5代细胞制备的饲养层细胞能够支持胚胎干细胞SF1-G呈边界清晰的克隆样生长。染色体核型检测SF1-G保持正常核型,碱性磷酸酶、表面标志物检测均呈阳性。实验建立了有效的小鼠胚胎成纤维细胞分离培养体系,并制备供胚胎干细胞进行增殖培养饲养层细胞体系,能够在实验室对SF1-G细胞保持正常未分化状态培养。     

小鼠非同步胚胎移植技术

目的:比较小鼠非同步胚胎移植技术。方法:将小鼠不同发育阶段的着床前胚胎非同步移植到不同假孕受体小鼠的输卵管或子宫内,观察非同步胚胎移植对小鼠胚胎移植成功率和后代出生重的影响。结果:CD-1小鼠受精卵、2-细胞胚胎、桑椹胚和囊胚都可移植到假孕0.5d受体小鼠输卵管继续发育,妊娠受体在移植后19d出生小鼠,胚胎移植出生率分别为56.3%、62.5%、59.4%和58.3%。桑椹胚和囊胚也可移植到假孕2.5 d受体子宫继续发育,妊娠受体在移植后17 d出生小鼠胚胎移植出生率分别为57.5%和62.5%。囊胚移植假孕0.5 d受体输卵管的出生小鼠的出生重显著大于移植假孕2.5 d受体子宫的出生小鼠。结论:不同发育阶段的胚胎若移植相同的假孕受体,则妊娠受体有相同的妊娠期;相同发育阶段的胚胎若移植不同的假孕受体,则妊娠受体有不同的妊娠期,假孕时间短的受体有较长的妊娠期和显著增加的小鼠出生重。     

非优质胚胎继续囊胚培养与冻融囊胚移植的临床应用价值

目的探讨将非优质胚胎继续囊胚培养与冻融囊胚移植的临床应用价值。方法收集生殖中心235个体外受精-胚胎移植(IVF-ET)和卵胞浆内单精子注射-胚胎移植(ICSI-ET)治疗周期中受精后第3天(D3)移植、冷冻保存后剩余的1026枚非优质胚胎继续囊胚培养,观察优质囊胚的形成情况,并玻璃化冷冻保存,若患者在新鲜移植周期妊娠失败,则在下一周期把囊胚解冻移植,比较冻融囊胚与同时期冻融卵裂胚的种植率和临床妊娠率。结果 D3非优质胚胎的优质囊胚形成率为36.6%,其中有43名患者的77枚优质囊胚在下一周期解冻移植,胚胎种植率(41.6%)和临床妊娠率(65.1%)显著高于同时期100名患者的202枚卵裂胚解冻后移植的胚胎种植率(25.3%)和临床妊娠率(43.6%)(P0.05),流产率无显著性差异(P0.05)。结论 1继续囊胚培养能有效筛选出非优质胚胎中具有发育潜能的胚胎,提高其利用率;2冻融囊胚移植能提高胚胎种植率和临床妊娠率。     

A comparative approach to somatic cell nuclear transfer in the rhesus monkey

BACKGROUND: Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. METHODS: A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. RESULTS: An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. CONCLUSIONS: The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.     

Reprogramming of a melanoma genome by nuclear transplantation

We have used nuclear transplantation to test whether the reprogramming activity of oocytes can reestablish developmental pluripotency of malignant cancer cells. We show here that the nuclei of leukemia, lymphoma, and breast cancer cells could support normal preimplantation development to the blastocyst stage but failed to produce embryonic stem (ES) cells. However, a blastocyst cloned from a RAS-inducible melanoma nucleus gave rise to ES cells with the potential to differentiate into multiple cell types in vivo including melanocytes, lymphocytes, and fibroblasts. Chimeras produced from these ES cells developed cancer with higher penetrance, shorter latency, and an expanded tumor spectrum when compared with the donor mouse model. These results demonstrate that the secondary changes of a melanoma nucleus are compatible with a broad developmental potential but predispose mice to melanomas and other malignant tumors on reactivation of RAS. Our findings serve as a paradigm for studying the tumorigenic effect of a given cancer genome in the context of a whole animal.     

利用亮甲酚蓝染色筛选优质猪卵母细胞

目的 利用亮甲酚蓝(BCB)染色提高猪体外胚胎培养体系的效率.方法 对猪卵丘卵母细胞复合体(COCs)进行BCB染色,观察染色后猪卵母细胞成熟率和早期胚胎发育能力并进行分析. 结果 着色组(BCB+)猪卵母细胞达到减数分裂Ⅱ(MⅡ)期的比率(91.6%)显著高于未着色组(BCB-)(64.0%)和对照组猪卵母细胞(77.5%)(P<0.05);BCB+孤雌猪胚胎的囊胚率(34.9%)显著高于BCB-(9.5%)和对照组(23.1%)(P<0.05);BCB+核移植猪胚胎的囊胚率(23.0%)显著高于BCB-(5.0%)和对照组(14.1%)(P<0.05).结论 BCB染色是一种有效筛选具有较强发育能力猪卵母细胞的方法.     

Derivation of cloned human blastocysts by histone deacetylase inhibitor treatment after somatic cell nuclear transfer with β-thalassemia fibroblasts

Derivation of embryonic stem cells from patient-specific cloned blastocysts by somatic cell nuclear transfer (SCNT) holds promise for both regenerative medicine and cell-based drug discovery. However, the efficiency of blastocyst formation after human SCNT is very low. The developmental competence of SCNT embryos has been previously demonstrated in several species to be enhanced by treatment with histone deacetylase inhibitors, such as trichostatin A (TSA), to increase histone acetylation. In this study, we report that treatment of SCNT embryos with 5 nM TSA for 10 h following activation incubation increased the developmental competence of human SCNT embryos constructed from β-thalassemia fibroblast cells. The efficiency of blastocyst formation from SCNT human embryos treated with TSA was approximately 2 times greater than that from untreated embryos. Cloned blastocysts were confirmed to be generated through SCNT by DNA and mitochondrial DNA fingerprinting analyses. Further, treatment of SCNT embryos with TSA improved the acetylation of histone H3 at lysine 9 in a manner similar to that observed in in vitro fertilized embryos.     

Cloned human embryonic stem cells for tissue repair and transplantation

One approach to overcome transplant rejection of human embryonic stem (ES) cells is to derive ES cells from nuclear transfer of the patient’s own cells. Because an efficient protocol for human somatic cell nuclear transfer (SCNT) has not been reported, several critical factors need to be determined and optimized. Our experience with domestic animals indicate that reprogramming time (the period of time between cell fusion and oocyte activation), activation method and in vitro culture conditions each play a critical role in chromatin remodeling and the developmental competence of SCNT embryos. In this review, we describe the optimization of human SCNT and derivation of human cloned ES cells. In our study, about approx 25% of human reconstructed embryos developed into blastocysts when we allowed 2 h for reprogramming to support proper embryonic development. Since sperm-mediated activation is absent in SCNT, an artificial stimulus is needed to initiate embryo development. Incubation with 10 μM calcium ionophore for 5 min followed by incubation with 2.0 μM 6-dimethyl amino purine was found to be the most efficient chemical activation protocol for SCNT using human oocytes. In order to overcome inefficiencies in embryo culture, we prepared human modified synthetic oviductal fluid with amino acids (hmSOFaa) by supplementing mSOFaa with human serum albumin and fructose instead of bovine serum albumin and glucose, respectively. Culturing human SCNT-derived embryos in G1.2 medium for the first 48 h followed by hmSOFaa medium produced more blastocysts than culturing in G1.2 medium for the first 48 h followed by culture in G2.2 medium or culturing continuously in hmSOFaa medium. The protocol described here produced cloned blastocysts at rates of 19–29%, which is comparable with the rates in cattle (approx 25%) and pigs (approx 26%) using established SCNT methods. A total of 30 SCNT-derived blastocysts were cultured, 20 inner cell masses (ICMs) were isolated by immunosurgical removal of the trophoblast, and one human cloned ES cell line (SCNT-hES1) with typical ES cell morphology and pluripotency was derived. Our approach opens the door for the use of autologous cells derived from nuclear transfer ES (ntES)-derived cells in transplantation medicine.     

骨桥蛋白对小鼠体外受精及早期胚胎发育的影响

目的 探讨骨桥蛋白（OPN）对小鼠体外受精及早期胚胎发育的影响。方法 120只昆明雌鼠和30只雄鼠,采用体外受精、胚胎培养方法,将小鼠精子、卵子、原核期胚胎和2-细胞期胚胎分别用不同浓度的OPN抗体预处理,观察小鼠受精、卵裂以及早期胚胎发育情况。结果 不同浓度的OPN抗体分别预处理精子和卵子后,与对照组比较,受精率显著降低(P＜0.01)。OPN抗体预处理原核期胚胎后,0.01mg/L OPN抗体组的卵裂率较对照组低,但差异无显著性 (P =0.052),1.00mg/L OPN抗体组的卵裂率低于0.01mg/L 组（P ＜0.01）及0.10mg/L组（P ＜0.05）。不同浓度的OPN抗体预处理2-细胞胚胎后可抑制胚胎发育。1.00mg/L OPN抗体组的4-细胞率和8-细胞率与0.10mg/L OPN抗体组相比差异无显著性(P ＞0.05),但前者的囊胚形成率显著低于后者（P ＜0.01）。1.00mg/L OPN抗体组的4-细胞率、8-细胞率以及囊胚形成率均显著低于0.01mg/L OPN抗体组（P ＜0.01）。结论 OPN可促进小鼠的受精和早期胚胎发育。     

四倍体胚胎发育阶段对胚胎干细胞嵌合体小鼠制备的影响

目的 探讨四倍体胚胎发育阶段对胚胎干细胞(ES)嵌合体小鼠制备的影响.方法 通过2-细胞胚胎电融合法制备四倍体胚胎,采用显微注射方法将ES细胞分别注入1-细胞、4-细胞、囊胚3个发育阶段的四倍体胚胎中.所用ES细胞分别为杂交系B6D2F1×129/Sv和近交系C57BL/6J,经胚胎移植和剖腹产以获得ES小鼠.结果 实...     

Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase

Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.     

Telomere Elongation Facilitated by Trichostatin A in Cloned Embryos and Pigs by Somatic Cell Nuclear Transfer

Telomere attrition and genomic instability are associated with organism aging. Concerns still exist regarding telomere length resetting in cloned embryos and ntES cells, and possibilities of premature aging of cloned animals achieved by somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), a histone deacetylase inhibitor, effectively improves the developmental competence of cloned embryos and animals, and recently contributes to successful generation of human ntES cells by SCNT. To test the function of TSA on resetting telomere length, we analyzed telomeres in cloned blastocysts and pigs following treatment of SCNT embryos with TSA. Here, we show that telomeres of cloned pigs generated by standard SCNT methods are not effectively restored, compared with those of donor cells, however TSA significantly increases telomere lengths in cloned pigs. Telomeres elongate in cloned porcine embryos during early cleavage from one-cell to four-cell stages. Notably, TSA facilitates telomere lengthening of cloned embryos mainly at morula-blastocyst stages. Knockdown of pTert by shRNA in donor cells reduces telomerase activity in cloned blastocysts but does not abrogate telomere elongation in the TSA-treated embryos (p?>?0.05). However, genes associated with recombination or telomerase-independent mechanism of alternative lengthening of telomeres (ALT) Rad50 and BLM show increased expression in TSA-treated embryos. These data suggest that TSA may promote telomere elongation of cloned porcine embryos by ALT. Together, TSA can elongate telomeres in cloned embryos and piglets, and this could be one of the mechanisms underlying improved development of cloned embryos and animals treated with TSA.     

Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling

BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming. METHODS: NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling. RESULTS: Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming. CONCLUSIONS: Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.