Serial nuclear transfer improves the development of interspecies reconstructed giant panda (Aluropoda melanoleuca) embryos

Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonquiescent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blastomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial II and serial III were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups were 19.4%, 13.5% and 10.3%, respectively, which are significantly higher than that in somatic NT group. These results indicate that the nuclei from nonquiescent somatic cells can support early development of reconstructed embryos and serial NT can improve the development rate of interspecies reconstructed embryos. These authors contributed equally to this work.     

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.     

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…     

Molecular genetics: DNA analysis of a putative dog clone

In August 2005, Lee et al. reported the first cloning of a domestic dog from adult somatic cells. This putative dog clone was the result of somatic-cell nuclear transfer from a fibroblast cell of a three-year-old male Afghan hound into a donor oocyte provided by a dog of mixed breed. In light of recent concerns regarding the creation of cloned human cell lines from the same institution, we have undertaken an independent test to determine the validity of the claims made by Lee et al..     

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.     

Dogs cloned from adult somatic cells

Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.     

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.     

Nuclear reprogramming and pluripotency

The cloning of mammals from differentiated donor cells has refuted the old dogma that development is an irreversible process. It has demonstrated that the oocyte can reprogramme an adult nucleus into an embryonic state that can direct development of a new organism. The prospect of deriving patient-specific embryonic stem cells by nuclear transfer underscores the potential use of this technology in regenerative medicine. The future challenge will be to study alternatives to nuclear transfer in order to recapitulate reprogramming in a Petri dish without the use of oocytes.     

Pregnancy: a cloned horse born to its dam twin

Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological recognition of fetal antigens influences the well-being of the fetus and the outcome of the pregnancy.     

Genotyping of Rhesus SCNT pluripotent stem cell lines

Somatic cell nuclear transfer (SCNT) into enucleated oocytes has emerged as a technique that can be used to derive mouse embryonic stem cell lines with defined genotypes. In this issue Byrne et al. report the derivation of two SCNT Rhesus macaca male stem cell lines designated CRES-1 and CRES-2. Molecular studies detailed in their paper provides supporting evidence that the chromosome complement of CRES-1 and CRES-2 was genetically identical to the male cell donor nucleus and that the mitochondrial DNA originated from different recipient oocytes. In this validation paper, we independently confirm that both stem cell lines were indeed derived by SCNT.     

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.     

Developmental reprogramming after chromosome transfer into mitotic mouse zygotes

Until now, animal cloning and the production of embryonic stem cell lines by somatic cell nuclear transfer have relied on introducing nuclei into meiotic oocytes. In contrast, attempts at somatic cell nuclear transfer into fertilized interphase zygotes have failed. As a result, it has generally been assumed that unfertilized human oocytes will be required for the generation of tailored human embryonic stem cell lines from patients by somatic cell nuclear transfer. Here we report, however, that, unlike interphase zygotes, mouse zygotes temporarily arrested in mitosis can support somatic cell reprogramming, the production of embryonic stem cell lines and the full-term development of cloned animals. Thus, human zygotes and perhaps human embryonic blastomeres may be useful supplements to human oocytes for the creation of patient-derived human embryonic stem cells.     

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％）．     

Somatic cell nuclear transfer

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.     

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.     

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.     

Application of a nuclear localization signal gene in transgene mice

Efficient gene transfer by cytoplasm co-injection will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine α-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.     

体外受精过程中多精受精发生机制

对哺乳动物体外受精(IVF)过程中多精受精的发生原因进行了阐述,指出多精受精的发生是影响各种哺乳动物体外受精效率的重要原因,其中在猪和人的IVF中表现得尤为突出。大量研究表明,除了物种的特异性以外,不完全的卵母细胞胞质成熟、异常透明带、高精子浓度、受精培养基中不适当的添加物以及在受精过程中的其他一些异常因素都与多精受精密切相关。