Comparison of efficiency of in vitro cloned sheep embryo production by conventional somatic cell nuclear transfer and handmade cloning technique

Conventional somatic cell nuclear transfer (SCNT) technique of in vitro production of cloned embryos involves use of costly and complicated micromanipulators. Handmade cloning (HMC) technique has been applied as efficient and cost‐effective alternative in many livestock species. The aim of the present study was to compare the efficiency of in vitro production and in vitro development of cloned sheep embryos by the two techniques. Cloned embryos were produced by conventional SCNT using micromanipulator apparatus and by HMC technique. Enucleation efficiency and efficiency of fusion with somatic cell (nucleus donor) were compared. Cleavage percentage was observed on day 2 of in vitro culture (IVC), and morula and blastocyst percentages were calculated on day 7 of IVC. Higher enucleation efficiency (96.98 ± 1.01 vs. 93.62 ± 1.03; p > .05) as well as fusion efficiency was obtained with HMC technique than with conventional SCNT (96.26 ± 1.34 vs. 92.63 ± 0.70, p < .05); 181 cloned sheep embryos were produced in vitro by conventional SCNT and 92 by HMC. Cleavage percentage observed on day 2 of in vitro culture was higher in HMC than SCNT (66.92 ± 3.72 vs. 55.97 ± 2.5, respectively, p < .05). Morula percentage obtained was higher in SCNT than HMC (44.12 ± 2.93 vs. 30.43 ± 6.79, respectively, p < .05), whereas blastocyst percentage obtained by HMC was higher (12.46 ± 4.96) than SCNT (5.31 ± 2.25; p > .05). It was inferred that HMC technique provides a cost‐effective and efficient method of in vitro production of cloned sheep embryos with a comparatively simpler technique with a possibility of automation. Efficiency of cloned embryo production could be improved further by propagating and standardizing this technique.     

体细胞克隆猪的研究与展望

用体细胞核移植的方法生产的克隆猪诞生至今已近5年，虽然同胚胎细胞核移植相比，体细胞核移植技术难度大，成功率低，但近年来用体细胞核移植技术克隆猪有了更深入的研究，在体细胞核移植基本机理和关键技术上取得了一定的进展。综述了用核移植的方法生产体细胞克隆猪的技术关键和难点及应用情况，并对提高猪体细胞核移植效率的策略进行了分析。     

水牛徒手克隆胚胎培养及冷冻条件的优化

本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养;采用了40% EG(ethylene glycol,EG)、25% EG＋25% DMSO(dimethylsulphoxide,DMSO) 和20% EG＋20% DMSO＋0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻;并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率。结果表明,微穴系统的卵裂率要显著高于平板系统(P<0.05),极显著高于微滴系统(P<0.01);且微穴系统的囊胚率(40.0%)极显著高于平板(19.8%)和微滴系统(8.3%)(P<0.01)。采用20% EG＋20% DMSO＋0.5 mol/L蔗糖作为冷冻保护剂时HMC囊胚存活率极显著高于40% EG(P<0.01);HMC重组胚的融合率和囊胚率均高于传统核移植法(P<0.05;P<0.01),而HMC囊胚的冷冻存活率与传统核移植生产的囊胚没有显著差异。以上结果说明水牛HMC可以替代传统核移植法生产克隆胚胎,微穴体系最适合水牛HMC胚胎的体外培养,且采用20% EG＋20% DMSO＋0.5 mol/L蔗糖对HMC囊胚进行玻璃化冷冻可以取得良好的冷冻效果。     

延边黄牛体细胞核移植的研究进展

牛体细胞核移植(somatic cell nuclear transfer,SCNT)是一套极其复杂的技术体系,它包括卵母细胞的成熟、供核细胞的准备、卵母细胞的显微操作、细胞融合、卵母细胞激活和胚胎培养。因此,许多因素影响着核移植胚胎发育。虽然陆续有克隆牛出生的报道,但是克隆效率依然低下。本综述对延边黄牛体细胞核移植体系的6个方面进行简单综述,以期为探究延边黄牛体细胞克隆的最优化条件、建立最优化的培养体系、提高克隆效率提供参考。     

Research Advances on Somatic Cell Nuclear Transfer in Yanbian Yellow Cattle

Bovine somatic cell nuclear transfer (SCNT) is a sophisticated technique system,including oocyte maturation,donor cell preparation and oocytes microscopic operation,fusion,activation and culture.Although the birth of cloning cattle has been reported recently,the efficiency of somatic cell cloning has remained lowly.In order to establish the optimization somatic cell nuclear transfer system of Yanbian Yellow cattle,this review summarized only from 6 main aspects mentioned above in this field.     

水牛徒手克隆的初步研究

徒手克隆是近年发展起来的一种手工化的核移植技术。本试验探讨了不同的去核方法、融合方法和供体细胞类型对水牛HMC效率的影响。试验结果表明,CB处理法去核与荧光染色法去核相比,去核效率及之后的重组胚发育能力均无显著差异(P＞0.05);一步法的融合率极显著高于两步法(P＜0.01),而两种方法构建的重组胚发育能力则无显著差异(P＞0.05);利用卵丘细胞所构建的HMC胚胎的融合率极显著高于耳皮肤成纤维细胞(P＜0.01),但重组胚的发育效果差异不显著(P＞0.05)。说明利用卵丘细胞作为HMC的供体细胞,采用CB处理法去核和一步融合的方法进行徒手克隆,其生产核移植胚胎效果较好。     

提高哺乳动物克隆效率的研究进展

体细胞核移植技术（somatic cell nuclear transfer,SCNT）又称为体细胞克隆技术,在生物医学、遗传修饰动物研究及大家畜的育种和良种扩繁等领域具有重要的理论意义和现实价值,但目前该项技术依然存在如克隆效率低下、克隆动物表型异常等问题。近年来,研究人员通过在培养基中添加小分子药物、选择优势供体细胞系（包括iPS细胞）、优化传统核移植参数、对发育关键基因（如XIST及H3K9me3去甲基化酶等）的靶向遗传调控等途径,探索提高克隆效率的新思路、新工艺和新方法,作者着重对上述研究进展进行简要综述。     

哺乳动物体细胞核移植胚胎发育中表观重编程的研究进展

体细胞核移植（somatic cell nuclear transfer,SCNT）是一种能将已分化的体细胞重编程为全能胚胎的繁殖生物技术,在良种扩繁、濒危物种保护和治疗性克隆等方面有着广泛的应用前景,但极低的克隆效率、克隆动物胎盘异常、出生后胎儿畸形等严重限制了该技术的实际应用。造成克隆效率低和胚胎发育异常的主要原因是供体核表观遗传重编程错误或不完全。1958年,将非洲爪蟾（Xenopus laevis）幼体肠细胞核移入去核卵母细胞,获得了第1例SCNT动物个体;1986年,通过电融合1个卵裂球与去核卵母细胞成功获得了3只存活的羔羊;1997年,将成年母羊的乳腺上皮细胞与去核卵细胞电融合,获得首个SCNT哺乳动物"多利",开启了克隆时代,目前牛、小鼠、山羊、猪、欧洲盘羊、家兔、家猫、马、大鼠、骡子、狗、雪貂、狼、水牛、红鹿、单峰骆驼、食蟹猴等相继成功克隆,其中最引人瞩目的是2018年食蟹猴的成功克隆。作者通过将SCNT胚胎与受精胚胎的发育进行对比,阐述了SCNT过程中DNA甲基化、组蛋白修饰、基因组印迹、染色体状态等的重编程过程和缺陷,并从表观修饰剂、组蛋白去甲基化酶、抑制Xist表达、补充鱼精蛋白和精子RNA方面探讨单独或联合消除表观遗传重编程障碍对克隆效率的影响。随着低样本量测序技术的发展和完善,人们能够在SCNT胚胎中检测到更详细的全基因组表观遗传修饰图谱,进一步揭示SCNT胚胎表观遗传重编程中的缺陷,为提高克隆效率提供了线索。通过上述内容的阐述,希望为后续开发联合消除多种表观遗传障碍而提高克隆效率的策略和思路。     

影响猪体细胞核移植效果的因素

克隆技术的研究已经取得了丰硕的成果,但猪体细胞核移植还受到众多因素的制约,克隆效率仍然较低,文章针对猪体细胞核移植的各项技术环节及影响因素进行探讨与分析,以期加快猪体细胞核移植技术的研究,提高猪体细胞核移植效率,进一步促进猪体细胞核移植技术的发展和运用。     

关于用动物属间体细胞核移植拯救濒临灭绝动物的研究

和克隆啮齿动物和家畜有所不同,由于濒危动物的卵母细胞的缺乏,克隆将要灭绝的物种需要具有选择性的方法，比如种属间体细胞核移植。文章以将喜马拉雅班羚体细胞注入牛卵母细胞的属间核移植后的囊胚的产生为例^[1]，介绍属间核移植的具体方法.由于将要灭绝的物种总数急剧减少，并且对它们实施人工繁殖技术的报道很少。因此,人工繁殖技术、属间和种内的体细胞核移植对增加有限的濒临灭绝物种的总体和保存这个物种是一个很有潜力的办法.希望这个方法会对环境保护和濒危动物的繁衍产生一定的作用.     

Developmental competence of HMC(TM) derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells

To enable us to handle a large number of oocytes at a given time and to have an increased throughput of cloned embryos, we attempted the Handmade cloning (HMC) technique, a zona-free method of bovine somatic cell nuclear transfer. Our objective was to study the developmental competence of the HMC derived embryos obtained using different types of somatic cells. A total of 6,874 cumulus-oocyte-complexes were used with either 7th or 11th passage fibroblasts (1st and 2nd groups, respectively), which were prepared from male animals, or granulosa cells (3rd group) as nuclei donors. The average cleavage rate was 65%, accompanied by a blastocyst rate of just 2% for the cleaved products and 5% for the >8-cell embryos, and there was no significant difference between the three groups. Out of 27 blastocysts recovered, 22 blastocysts were transferred to 22 recipients, resulting in two pregnancies. One pregnancy was lost after the fourth week while the other progressed to full term with the birth of a male calf. This first successful cloning of a male calf with the HMC technique in Europe indicates the successful adoption and establishment of this technique in our laboratory, and that this technique can be successful in producing viable embryos.     

徒手克隆工具自制及改进

徒手克隆技术(handmade somatic cell cloning,HMC)解决了由于昂贵设备对核移植技术应用的制约问题。通过徒手克隆刀的自制,不但进一步节省了成本,且更能符合使用者的习惯,提高总的切割效率。试验结果表明,利用自制克隆刀进行半卵法去核,可以达到每小时切割496枚卵母细胞的切割速度和88.14%的存活率;自制聚集针具有制作简单,可以打磨、效果好等特点,可以进一步简化和降低徒手克隆技术的成本,提高克隆效率。     

Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage

The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer.     

Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre‐implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.     

Improving the Quality of Miniature Pig Somatic Cell Nuclear Transfer Blastocysts: Aggregation of SCNT Embryos at the Four‐cell Stage

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1‐positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four‐cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four‐cell stage improves the total number of nuclei and the percentage of POU5F1‐positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four‐cell stage did not exhibit a decrease in the percentage of POU5F1‐positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four‐cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1‐positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers.     

Effect of donor cell type on nuclear remodelling in rabbit somatic cell nuclear transfer embryos

Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.     

Epigenetic Regulation of Foetal Development in Nuclear Transfer Animal Models

Somatic cell nuclear transfer (SCNT, 'cloning') holds great potential for agricultural applications, generation of medical model animals, transgenic farm animals or by 'therapeutic cloning' for generating human embryonic stem cells for the treatment of human diseases. However, the low survival rate of SCNT-derived pregnancies represents a serious limitation of the current technology. In order to overcome this hurdle, a deeper understanding of the epigenetic reprogramming of the somatic cell nuclei and its effect on the pregnancy is needed. Here we review the literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodelling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which the body of the foetus is formed, seems to be highly efficient. Defects in the extra-embryonic tissues are probably the major cause of the low success rate of reproductive cloning. Methods to partially overcome such problems exist, yet more future research is needed to find practical and efficient methods to remedy this problem. Improvement of the survival of foetuses is a central issue for the future of agricultural SCNT not only for its economic viability, but also because in lack of improvements in animal welfare current regulations can block the use of the method in the EU and several other countries.     

Recent progress in bovine somatic cell nuclear transfer

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full‐term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT.     

Effect of calf death loss on cloned cattle herd derived from somatic cell nuclear transfer: Clones with congenital defects would be removed by the death loss

To increase public understanding on cloned cattle derived from somatic cell nuclear transfer (SCNT), the present review describes the effect of calf death loss on an SCNT cattle herd. The incidence of death loss in SCNT cattle surviving more than 200 days reached the same level as that in conventionally bred cattle. This process could be considered as removal of SCNT cattle with congenital defects caused by calf death loss. As a result of comparative studies of SCNT cattle and conventionally bred cattle, the substantial equivalences in animal health status, milk and meat productive performance have been confirmed. Both sexes of SCNT cattle surviving to adulthood were fertile and their reproductive performance, including efficiency of progeny production, was the same as that in conventionally bred cattle. The presence of substantial equivalence between their progeny and conventionally bred cattle also existed. Despite these scientific findings, the commercial use of food products derived from SCNT cattle and their progeny has not been allowed by governments for reasons including the lack of public acceptance of these products and the low efficiency of animal SCNT. To overcome this situation, communication of the low risk of SCNT technology and research to improve SCNT efficiency are required.