A comparison of established and new approaches in ovine and bovine nuclear transfer

Several breakthroughs in nuclear transfer research were first achieved in sheep, although cattle soon became the main livestock species of interest. However, sheep still offer significant advantages both in basic and applied research. With increased interest in cloning of livestock, new approaches have been developed for both sheep and cattle nuclear transfer technology. These include methods for zona-free nuclear transfer that can be performed with or without the use of micromanipulator. Here we describe four different nuclear transfer methods including the traditional micromanipulation-assisted method in sheep, zona-free method in sheep in which the order of enucleation and nucleus delivery have been reversed ("reverse-order" cloning) and zona free manual cloning methods ("hand-made cloning") for embryonic and somatic cloning in cattle. The purpose of this paper is to encourage people to familiarize themselves with these different methods available and to help them choose and test the method most suitable for their particular circumstances.     

Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.     

Simple, fast, and efficient method of manual oocyte enucleation using a pulled Pasteur pipette

Cloning mammals by somatic cell nuclear transfer entails the replacement of oocyte chromosomes with the nucleus of a somatic cell. A major step in this technique is to efficiently produce large batches of enucleated oocytes, a process that requires considerable micromanipulation skills and expensive equipments. Here, a simple, fast, and efficient method of manual oocyte enucleation was introduced that can be adopted in every laboratory with the minimum equipments. Common laboratory glass pipettes were pulled on the flame of a burner and then used for manual bisection or enucleation of sheep and goat zona-free oocytes by passing them through the discontinuous cutting border of culture medium and mineral oil. The described techniques showed a certain efficiency to conveniently bisect or enucleate large batches of sheep, and goat oocytes being pre-treated with demecolcine. The method may be straightforward for simple manipulation of oocytes of other species and for development of automated cloning methods as well.     

Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs

Handmade cloning (HMC) is now an established procedure used in several species for somatic cell nuclear transfer, but only applied in two related laboratories for pigs. The aim of this review is to facilitate widespread application by summarizing the process of establishment and explaining the background of the incorporated special approaches. Optimized steps of traditional cloning in pigs (in vitro maturation, activation, embryo culture) were merged with those of the micromanipulation-free HMC that has been modified according to the specific needs of sensitive porcine oocytes (partial zona digestion before enucleation, two-step zona-free fusion with the somatic cell; initiation of activation with the second fusion). The zona-free approach required embryo culture to the blastocyst stage before surgical transfer of embryos to the uterine horns of recipient sows in the proper phase of an unstimulated cycle. Eventually a competitive, inexpensive and reliable alternative to traditional porcine nuclear transfer cloning techniques evolved that is also suitable to produce transgenic offspring containing various genetic modifications to establish models for several human diseases with genetic background. Further improvements and involvement of additional techniques to increase the overall efficiency and facilitate practical applications are expected in the foreseeable future.     

Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation

The purpose of our work was to establish an efficient protocol for activation of porcine cytoplast-fibroblast constructs produced by the handmade cloning technique. Firstly, we investigated a combined electrical and chemical activation protocol for parthenogenetic development of in vitro matured zona-free oocytes. Oocytes were activated by one 80 micros pulse and subsequently cultured in cytochalasin B and cycloheximide. Developmental rates of blastocysts from activated oocytes were 49+/-1 and 40+/-2%, when using one 80 micros pulse of 0.85 or 1.25 kV/cm, respectively. The activation procedure was further confirmed by a simultaneous re-fusion and activation of bisected oocytes, resulting in a blastocyst rate of 41+/-8%. Secondly, the activation protocol was applied in the handmade cloning technique. In vitro matured zona-free porcine oocytes were bisected and halves containing no chromatin, i.e. the cytoplasts, were selected. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused to one fibroblast by one 80 micros pulse of 1.25 kV/cm. After 1h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously by one 80 micros pulse of 0.85 kV/cm, and subsequently cultured in cytochalasin B and cycloheximide. The development of reconstructed embryos to the blastocyst stage was in average 21+/-4%, and total blastocyst cell counts were in average 48+/-3. Thus, the combined electrical and chemical activation procedure resulted in efficient blastocyst development in the handmade cloning technique.     

Cloned cattle derived from a novel zona-free embryo reconstruction system

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.     

克隆动物研究进展

本文介绍了世界上和中国采用细胞核移植技术克隆动物的研究历史。综述了细胞核移植的程序、方法和影响因素,包括受体卵母细胞的去核、供体细胞核的制备、核移植、激活、受体细胞与供体细胞的融合、重组胚的体内和体外培养以及胚胎移植产生克隆动物。对克隆动物研究和应用前景进行了讨论。近期的研究结果表明,多代克隆可产生大量遗传性相同的动物,不久的将来克隆技术在商业上的应用将成为现实。     

Development of a zona-free method of nuclear transfer in the mouse

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.     

Effect of timing of the removal of oocyte chromosomes before or after injection of somatic nucleus on development of NT embryos

Cloning methods are now well described and becoming routine. Yet the frequency at which live cloned offspring are produced (as a percentage of starting one-cell embryos) remains below 5% irrespective of nucleus donor species or cell type. In considering the cause(s) of this universally low efficiency, features common to all cloning protocols are strong candidates. One such shared feature is enucleation; the donor nucleus is inserted into an enucleated cytoplast (ooplast). However, it is not known whether a nucleus-free metaphase II oocyte is developmentally impaired other than by virtue of lacking chromosomes, or if in nuclear transfer protocols, enucleation removes factors necessary to reprogram the incoming nucleus. We have here investigated the role of enucleation in nuclear transfer. Three hours after the injection of cumulus cell nuclei into non-enucleated oocytes, 65% contained two distinct metaphase spindles, with the remainder exhibiting a single spindle in which oocyte-derived and nucleus donor chromosomes were mixed. However, staining only one hour after donor nucleus insertion revealed that most had two discrete spindles. In the absence of staining, the donor nucleus spindle was not visible. This provided a straightforward way to identify and select the oocyte-derived metaphase chromosomes 1 h after donor nucleus microinjection, and 34-41% cloned embryo developed to the morulla-blastocyst stage following Sr(2+)-induced activation. Of these, two (1% of starting one-cell embryos) developed to term, an efficiency which is comparable to that obtained for controls (6 clone; 1-2%) in which enucleation preceded nuclear transfer. In conclusion, the timing of the removal of oocyte chromosomes before or after injection of somatic nucleus had no effect on cloned embryo development. These findings argue that neither oocyte chromosome depletion per se, nor the potential removal of "reprogramming" factors during enucleation explain the low efficiency of nuclear transfer cloning.     

“Conservation cloning” of vulnerable Esfahan mouflon (Ovis orientalis isphahanica): in vitro and in vivo studies

Among the wide range of bio-conservational strategies envisaged, recent accomplishments in the field of interspecies somatic cell nuclear transfer (iSCNT) hold considerable promise due to its unique potential to decelerate or prevent rapid loss of animal genetic resources, and even to revive extinct species. Accordingly, this study was carried out to investigate if in vitro matured and enucleated oocytes of domestic sheep could be used for interspecies conservation cloning of Esfahan mouflon (Ovis orientalis isphahanica), a vulnerable species classified by the International Union for Conservation of Nature. Cryo-banked fibroblasts of a mouflon (derived from a genome resource bank) and a domestic sheep (prepared during a recent study) were cultured in vitro and used for karyotyping. Prior to SCNT, fibroblast donor cells were serum starved for 5 days. Using the zona-free SCNT technique, in vitro matured and enucleated domestic sheep oocytes were reconstituted with nuclei donor cells of mouflon and domestic sheep, and their competencies for in vitro development to the blastocyst stage were compared. The cloned mouflon blastocysts were then surgically transferred into the uterus of the synchronized domestic sheep. Karyotype analysis confirmed that fibroblasts of the Esfahan mouflon had the correct number of diploid chromosomes (2n = 54). Evaluation of 907 activated reconstructs [Esfahan mouflon (n = 667), domestic sheep (n = 240)] revealed no significant difference in the term of blastocyst development (7.6 ± 0.5% vs. 9.3 ± 0.5%, respectively). After the transfer of 12 cloned Esfahan mouflon blastocysts to five domestic sheep recipients, two (40.0%) pregnancies were established in which both (100%) were sustained until caesarean section (days 147 and 150 of pregnancy, respectively) and culminated in the live births of cloned Esfahan mouflon lambs. However, the newborns did not survive and died soon after birth. Karyotype and genetic analyses confirmed that both clones had correct diploid chromosome number (2n = 54), and were genetically identical to each other in addition to their original cell donor. This study highlighted the importance of “conservation cloning” using closely related abundant alternate species.

     

Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.     

Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer

The enucleation of oocytes to be used as host cytoplasts for embryo reconstruction by nuclear transfer is an important limiting step when cloning mammals. We propose an enucleation technique based on the removal of chromatin after oocyte activation, at the telophase stage, by aspirating the second polar body and surrounding cytoplasm. In a preliminary experiment to determine an optimal activation protocol, oocytes were matured for 26 and 30 hr and exposed for 5 min to 7% ethanol and/or for 3 hr at either 25 or 4°C. Relative to most activation treatments tested, oocytes matured for 30 hr and exposed to ethanol alone showed highest activation rates, as determined by low levels of H1 kinase activity within 90 min from exposure and high pronuclear formation (82%) after 12 hr of culture. No synergistic effect on activation rates was observed when oocytes also were exposed to reduced temperature after ethanol treatment. Microsurgical removal of the telophase-stage chromatin in a small volume of cytoplasm adjacent to the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes (98% versus 59%; P < 0.01). Moreover, compared with a nuclear transfer protocol based on enucleation of metaphase-arrested oocytes followed by aging and cooling, more (38% versus 16%; P < 0.001) and better-quality blastocytes (126 versus 84 nuclei per blastocyst; P < 0.02) were obtained from embryos reconstructed using the telophase procedure. Higher development potential of embryos reconstructed by the telophase procedure may be attributed to (1) the selection of oocytes that activate and respond by extruding the second polar body, (2) avoiding the use of DNA dyes and ultraviolet irradiation, and (3) the limited removal of cytoplasm during enucleation. The ease with which telophase enucleation can be performed is likely to render this technique widely useful for research and practice on mammalian cloning. Mol. Reprod. Dev. 49:29–36, 1998. © 1998 Wiley-Liss, Inc.     

Development of cloned embryos from adult rabbit fibroblasts: effect of activation treatment and donor cell preparation

This research was to study the in vitro and in vivo development of cloned embryos derived from adult rabbit fibroblasts following various activation protocols. Effects of serum starvation and passage number of donor cells on the efficiency of cloning were also examined. In experiment I, oocytes were activated either by electric pulses or by electric pulses followed by culture with 6-dimethylaminopurin (DMAP). For experiment II, the best activation protocol from experiment I was employed for cloning using adult rabbit fibroblasts that were cultured for 0-15 passages. In experiment III, the effect of serum starvation of the donor cells on cloning was examined. Finally, in experiment IV, embryo transfers were conducted. These experiments showed that combined electrical pulse and DMAP treatment resulted in superior parthenogenetic blastocyst development (up to 29%), and that activation of the cytoplast before versus after fusion was not different in supporting the in vitro development of nuclear transferred embryos (16%-18% blastocysts). Adult fibroblasts derived from nonpassaged cells were less capable of developing into blastocysts than passaged cells (6% vs. 17%). Serum starvation of donor cells improved cleavage (up to 71%) but did not improve blastocyst development (13%), and no progeny was obtained, irrespective of the treatment. Cell-cycle analysis of adult rabbit fibroblast cells showed that passage 6 and 12 cells were more likely to be in G(0)/G(1) than passage 0 cells, which agrees with the improved embryo development in the passaged-cell groups.     

完整的供体细胞膜及供体胞质对小鼠体细胞克隆胚发育的影响

利用显微注射和电融合的方法都可以成功地获得体细胞克隆小鼠, 由于电融合法操作耗时, 融合率低, 因而大多数克隆小鼠是采用注射方法。而注射法需要将供体细胞核从细胞中分离出来, 此分离操作有可能导致对DNA的损伤, 曾有人使用直径较粗的注射管进行完整的供体细胞注射, 这种方法操作相对简单而且对供体核没有损伤。为了研究这种方法在小鼠核移植中是否适用, 本实验使用完整的小鼠卵丘细胞作供体, 进行显微注射, 结果显示, 完整的卵丘细胞注入卵母细胞后, 无论在1小时或者6小时激活, 大部分的重构胚在2细胞期碎裂, 而去掉细胞膜的供体体细胞核注入卵母细胞后, 重构胚可以卵裂并进一步发育。卵母细胞去核后不注射供体也发生碎裂, 大部分的孤雌胚(不去核)在完整的卵丘细胞被注入后同样发生碎裂。在供体卵丘细胞刚破膜后即被注入卵胞质和供核被充分剥离后注入两种情况下获得的重构胚的体外发育中, 前者发育各期的比率显著低于后者。这些结果说明完整的卵丘细胞膜阻碍了卵胞质对体细胞核的重编程作用, 造成碎裂; 注入卵胞质的供体质膜和胞质成分影响了克隆胚的体外发育。     

Preparation of young preactivated oocytes with high enucleation efficiency for bovine nuclear transfer

To improve the enucleation rate in newly matured bovine oocytes, we investigated the position of cytoplasmic chromatin in relation to the polar body and the consequent enucleation efficiency before and after sequential activation with calcium ionophore A23187 and cycloheximide. Oocytes aspirated from the follicles of slaughterhouse-collected ovaries were cultured for 18 to 20 h. With Hoechst staining, only 40.7% of the chromatin material was found adjacent to the first polar body in metaphase II oocytes, while 100% was located adjacent to the second polar body in oocytes after the activation. Enucleation trials after activation showed a higher enucleation rate (91.5%) than that before activation (59.9%). The following experiment determined the effect of using both kinds of cytoplast on the in vitro development of nuclear transfer embryos. Blastomeres of the 32-cell-stage in vitro-produced embryos were transferred, fused to the activated cytoplasts and cultured in vitro. No significant difference was detected in fusion, cleavage or development to blastocysts obtained 7 d (174 h) post fusion. In conclusion, this study showed that young in vitro-matured bovine oocytes sequentially activated with calcium ionophore and cycloheximide have cytoplasmic chromatin material adjacent to the second polar body, leading to a high enucleation rate.     

Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer

Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.     

Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes

The present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two-, four-, or eight-cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0–91.6%), and activation (95.2–96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear-like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non-PCC (NPCC; 52.9%) embryos fused with the two-cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus-like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four- and eight-cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos. © 1994 Wiley-Liss, Inc.     

Cloned calves from chromatin remodeled in vitro

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.     

家畜体细胞克隆中核重编程机理研究进展

体细胞克隆在绵羊、山羊、牛、猪等家畜中获得了成功,但目前的克隆效率非常低。克隆效率低使家畜体细胞克隆技术在畜牧业生产及其他领域的应用受到极大的限制,问题的根源在于对体细胞克隆中核重编程的分子机理缺乏了解。供体细胞核移入去核的卵母细胞后,必须经过后成表观遗传修饰的重编程,从而恢复供体细胞核的全能性,才能保证重构胚的正常发育及个体的正常生长。本文从移植核的重构、DNA甲基化总体改变、组蛋白修饰、X染色体失活、端粒长度和端粒酶活性恢复、印迹基因及其他与发育相关基因的表达及核重编程的影响因素等几个方面探讨了体细胞克隆中的核重编程机理,为克隆效率提高的方法研究提供理论依据。