Embryonic stem cell as nuclear donor could promote in vitro development of the heterogeneous reconstructed embryo

The nucleus of a somatic cell could be dedifferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellucida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic stem cells was 16.1%, which was significantly higher than that of both the adult mouse fibroblast cells (0%-3.1%, P < 0.05) and fetus mouse fibroblast cells (2.1%-3.7%, P < 0.05). Chromosome analysis confirmed that blastocyst cells were derived from ES donor cell. These observations show that reprogramming is easier in interspecific embryos reconstructed with ES cells than that reconstructed with somatic cells, and that ES cells have the higher ability to direct the reconstructed embryos development normally than fibroblast cells.     

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

Somatic cell nuclear transfer has been succeeded in procedures of nuclear transfer. One is single nucleartransfer, the other is serial nuclear transfer. Viable animals have been cloned in different species using both me-thods[1—6]. Different nuclear recipients and donors wereused in serial nuclear transfer, namely, transferring thenuclear of reconstructed embryo into enucleated MⅡoocytes[7], transferring the nuclear of reconstructed em-bryos at one cell stage into enucleated zygote[4] and t…     

Nuclear transfer using nonquiescent adult fibroblasts from a bovine ear

The natural reproduction of mammal is sexual reproduction, which needs fertilization involving sperm and oocyte. Nuclear transfer provided an asexual reproduction method for mammal. Donor cells used in previous experiments of nuclear transfer were mostly from undifferentiated or non-terminally differentiated cells, such as embryonic or fetal cells. However, since Wilmutet al. obtained a viable lamb by transfer of an adult sheep somatic cell into an enucleated oocyte, nuclear transfer using adult somatic cell has been successful in several species. Wilmutet al. suggested that it was a key factor for the success of somatic nuclear transfer to induce the donor cells into GO phase (“GO-phase hypothesis”). In order to verify the Gophase hypothesis, nonquiescent adult fibroblasts from a bovine ear were transferred into enucleated bovine oocytes. The experiments showed that the rate of electrofusion after micromanipulation was above 50%, the cleaving rate was 54.5% and 9.1% of those reconstructed embryos developed to 32-cell stage. These results indicate that for cattle, nuclei from nonquiescent adult somatic cells introduced into enucleated oocytes are at least capable of supporting early development.     

Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse acti-vation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞，以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体，采用核移植方法，构楚了克隆胚胎．在同种克隆中，以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％，P〈0．05），1．8％的ES细胞克隆胚胎发育到囊胚阶段，而成纤维细胞克隆胚胎没能发育到囊胚阶段；在异种克隆中，以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

Green fluorescent protein （GFP） transsenic pig produced by somatic cell nuclear transfer

Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm animals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfecUon system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe- tal-derived fibroblast cells cultured with 4.0 μL/mL liposome and 1.6 μg/mL plasmid DNA for 6 h resulted in the highest transfecUon rate （3.6%）. The percentage of GFP reconstructed embryos that de- veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Reconstructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 delivered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.     

Production of transgenic blastocyst of sheep by somatic cell cloning

Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP- N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P＞0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.     

Clone of Chinese Jinan red-cross yellow cattle and evaluation of reproductive characteristics of cloned calf

Somatic cell clone technology is a viable approach to preserving endangered livestock and wildlife genetic resources. In the present research, somatic cell nuclear transfer （SCNT） was performed using granulose cells from the critical endangered Chinese red-cross yellow cattle as donor cells. A total of 211 oocytes were manipulated and 166 （79%） of them were successfully enucleated. 112 （67.4%） SCNT embryos were reconstructed, 94 （83%） of them cleaved, and 48 （43 %） of them developed to blastocyst stage. SCNT blastocysts were transferred to 6 Holstein recipients, and 2 （33%） of them were found to be pregnant. One of them maintained to term and delivered a calf, whereas another aborted. Effect of different fusion buffer （mannitol vs. Zimmerman fusion buffer） and different activation methods （calcium ionophore＋6-DMAP vs. cycloheximide＋CB） on fusion rate and development of SCNT embryos were investigated. The results indicated that： （i） on condition of two DC pulses of 2.5 kV/cm for 10 μs each, fusion rates were higher in mannitol solution than in Zimmerman fusion buffer （71% vs. 61%, respectively, p 〈 0.05）, but the blastocysts rates did not differ between two treatments （36 % vs. 39 %, p〉0.05 ）; （ii） There was no significant difference in development rates to the blastocyst stage for SCNT embryos activated by calcium ionophore＋6-DMAP or by cycloheximide＋CB （42% vs. 46%, respectively, p〉0.05）. Microsatellite DNA analysis examining 28 loci confirmed that the cloned calf was genetically identical to the donor Jinan red-cross yellow cattle and different from the recipient females. Growth and reproductive performance of cloned cow were evaluated, and there were no difference i cross-red n it between cloned and normal control Jinan yellow cattle. Furthermore, the cloned yellow cow has delivered a healthy yellow calf.     

卵母细胞老化对小鼠体细胞克隆胚发育的影响

就小鼠卵母细胞的卵龄对克隆胚的体外发育能力的影响进行了检测,以确定最佳的取卵时间,以及取卵后进行核移植操作的可耐受的时间。采用PMSG和hCG超排B6D2F1雌鼠。在体内老化实验中,分别于注射hCG后13,15,17,20h取卵用于核移植操作;在体外老化实验中,所有的卵均于注射hCG后13h取出并培养,然后在13,15,17,20h进行核移植操作。每个时间点的核移植操作在1h内完成,重构的胚胎培养1h后进行激活。结果显示:在体内老化实验中,注射hCG后13h取卵,获得的克隆囊胚发育率最高,为56.0%,15h取卵仍维持了其支持胚胎体外发育的能力,但17h及更长时间后取的卵,重构后的囊胚发育率显著下降(18.1%)。注射hCG后15h取的卵孤雌发育率最高(囊胚发育率为90.1%),并在17h仍维持较高的孤雌发育能力,在20h显著下降。在体外老化试验中,于注射hCG后13h取卵,分别于13,15,17h重构,都获得了较高的囊胚发育率(分别为57.7%,52.2%,46.3%),而在注射hCG后20h重构,囊胚发育率显著下降(14.8%)。体外老化的孤雌胚在注射hCG后22h激活时囊胚发育率显著下降。结果表明:卵母细胞在体内和体外老化都影响克隆胚的体外发育,最佳取卵时间为注射hCG后13h,取卵后立即用于重构可获得最佳囊胚发育率;体内、外老化卵支持重构胚较好发育的时间间隔是不同的,体内老化卵支持重构胚发育能力从注射hCG后17h开始下降,体外老化卵则发生在20h。这些结果可以指导操作,有利于在应用核移植进行其他基础研究时降低操作引起的误差。     

Microsatellite DNA analysis proves nucleus of interspecies reconstructed blastocyst coming from that of donor giant panda

A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g⁰¹⁰ was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.     

实验用小型猪胎儿成纤维细胞和骨髓间充质细胞为核供体生产转基因克隆胚胎

目的比较不同类型体细胞对生产转基因克隆胚胎效率的影响。方法利用脂质体介导的方法将质粒pEGFP-N1转染到五指山小型猪胎儿成纤维细胞和骨髓间充质细胞,经过G418筛选后均获得了阳性细胞株。然后分别以两种类型转基因细胞以及未转基因细胞为核供体进行体细胞核移植,比较不同类型供体细胞克隆胚胎的囊胚发育率。结果胎儿成纤维细胞和骨髓间充质细胞克隆胚的囊胚发育率差异不显著(P>0.05,8.3%vs.7.1%);转基因胎儿成纤维细胞(9.6%)和转基因骨髓间充质细胞(9.9%)克隆胚胎的囊胚发育率差异不显著(P>0.05,9.6%vs.9.9%);每一种类型供体细胞转基因与否对克隆胚的囊胚发育率无影响(P>0.05)。结论通过体细胞核移植技术,小型猪骨髓间充质细胞与胎儿成纤维细胞均可有效地生产转基因囊胚。     

Cloned pigs produced by nuclear transfer from adult somatic cells

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.     

小鼠精子微注射受精卵的体外发育与移植的研究

用显微注射方法,将精子注入小鼠卵母细胞卵周隙中,使精卵体外受精获得受精卵。并对精子注射后卵母细胞的成活率、受精率和受精卵的发育率以及移植后受胎率等方面进行了系统的研究,结果表明:卵子存活率为75.36％(1309/1737),其中264枚卵裂,受精率为20·17％(264/1309),卵裂卵经体外培养后有192枚发育到桑椹胚及胚泡期,发育率为72.73％(192/264)。将其中73枚(桑椹胚10枚,肛泡63枚)胚胎移植到10只假孕受体子宫中,一只受体妊娠产仔9只,仔鼠发育正常。     

Somatic cell bovine cloning: Effect of donor cell and recipients

Nuclear transfer (NT) is an efficient technique for assessing the developmental potential of a nucleus and for analyzing the interactions between the donor nucleus and the recipient cytoplasm. In amphibians, thought nuclei of adult kerationocytes support development to the juvenile, tadpole stage, no development to the adult stage was re- ported[1], leaving open the question of whether a different- tiated adult nucleus can be fully reprogrammed. The first cloned offspring developed from differ…     

兔ES样细胞系的建立及其特性分析

报道从２３７枚家兔胚胎中建成７个可连续传代的ＥＳ样细胞系。建系条件为，使用小鼠原始胚胎成纤维细胞（ＭＥ）作饲养层，以含１０％胎年血清和１０％兔血清的ＤＭＥＭ／Ｆ１２为培养基，添加白血病抑制因子（ＬＩＦ）或上皮生长因子（ＥＧＦ），胚龄为９０，９６ｈ。该细胞系的细胞。在许多方面类似于小鼠ＥＳ细胞，具干细胞的形态特征，呈集落型生长，可连续传代并保持其形态特征，具有一定的自发分化和诱导分化的能力，悬浮培养     

Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

Great progress have been made in animal cloning in China, as evidenced by the live births of cloned cat- tle[1,2], goats[3,4], and sheep[5]. In contrast, pig cloning is still in its infancy though limited fundamental studieshave been conducted[6]. It is g…     

Reconstruction of human embryos derived from somatic cells

Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into M Ⅱ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyteactivation and 2PN formation, we removed the female PN.By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation,and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into the blastocyst stage in vitro.     

Production of transgenic calves by somatic cellnuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.     

Effect of leptin on oocyte maturation and subsequent pregnancy rate of cloned embryos reconstructed by somatic cell nuclear transfer in pigs

Cloning pigs by somatic cell nuclear transfer （SCNT） has wide applications in basic research, human medicine and agricultural production. To improve cloning efficiency, the effect of two basic maturation media, NCSU-23 and TCM199, was compared, and TCM199 was selected for the following experiments with leptin. We systematically studied the effects of leptin supplementation on oocytes in vitro maturation （IVM）, in vitro development of parthenogenetically activated （PA） and SCNT embryos and in vivo development of SCNT embryos after embryo transfer （ET）. The results showed that supplementation of 100 or 200 ng/ml leptin into the maturation medium did not greatly affect nuclear maturation of oocytes, or cleavage rates of PA and SCNT （P 〉 0.05）. Blastocyst rates of PA and SCNT embryos were significantly improved when 100 or 200 ng/ml leptin was added to maturation medium, and the number of cells in PA blastocysts was also improved （P 〈 0.05）. The number of cells in blastocyst of SCNT was improved, when 100 ng/ml leptin was added （P 〈 0.05）. Furthermore, supplementation of 100 or 200 ng/ml leptin to the IVM medium may improve pregnancy rate and the delivery rate inpig cloning.     

牛体外受精胚胎成份明确培养系统的建立

以 SOF为基本培养液 ,分别添加血清、PVA以及同时添加 PVA、肌醇和柠檬酸钠 ,进行牛体外受精卵的发育培养 ,三个处理组的囊胚发育率分别为 :3 2 .6%、1 1 .9%和 1 5 .9% ,血清处理组极显著高于两个 PVA处理组 (p<0 .0 1 ) .在此基础上 ,将受精处理后的卵子去掉卵丘细胞后 ,培养于 SOF PVA 肌醇 柠檬酸钠培养液内 ,以未去掉卵丘细胞的卵子为对照 ,两个处理组的囊胚发育率分别为 1 0 .8%和 2 1 .1 % .二者间没有显著差异 .结果表明没有血清和卵丘细胞的成份明确的培养系统能够支持牛体外受精卵发育至囊胚阶段 .