Factors influencing the somatic cell nuclear transfer efficiency in pigs

Using a data set from our laboratory, we assessed the effects of several factors on pig cloning efficiency. The results demonstrated that cells at high confluence (>90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning efficiency (number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to 79% confluence and 80% to 89% confluence. Cells with four, five and six passages compromised the pregnancy and delivery rates compared with first passage cells. The number of blastocysts transferred by somatic cell nuclear transfer (SCNT) did not significantly affect the cloning efficiency, but transfer of blastocyst derived from in vitro culture 5 d after SCNT achieved a significantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig fibroblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT efficiency in pigs.     

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii) interspecific cloned embryos

The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT) was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.     

印记基因XIST和H19 DNA甲基化水平与猪克隆胚胎发育效率关联分析

目的 体细胞核移植(Somatic cell nuclear transfer,SCNT)在农业、生物医学等领域应用广泛,但是克隆效率太低制约了该技术的应用和推广。本研究的目的在于探究印记基因XIST和H19 DNA甲基化水平与克隆效率的联系。方法 利用同一头猪源耳朵成纤维细胞培养获得14个细胞克隆团,分别作为供体细胞进行SCNT试验,统计比较以各克隆团为供体细胞生产克隆胚胎的囊胚率以及各囊胚的XIST和H19基因调控区的DNA甲基化水平,对XIST和H19基因差异甲基化区域DNA甲基化水平与克隆胚胎的囊胚率进行相关性分析。结果 以1号克隆团为供体细胞得到囊胚的XIST基因DNA甲基化水平最高且囊胚率最低,分别为65.04%、8.6%,以14号克隆团为供体细胞得到XIST基因囊胚的DNA甲基化水平最低但囊胚率最高,分别为16.68%、38.2%;相关性分析表明XIST基因的DNA甲基化水平与囊胚率之间存在高度负相关(|r|=0.8125>0.8)。H19基因A侧甲基化平均水平最高和最低分别为5.12%和0.61%,相关性分析表明H19基因A侧DNA甲基化水平与囊胚率间只有极弱的相关(|r|=0.1647<0.3);H19基因G侧DNA甲基化水平最高和最低分别为90.92%和72.69%,相关性分析表明H19基因G侧DNA甲基化水平与囊胚率间只有低度相关(0.3<|r|=0.3098<0.5)。结论 XIST基因DNA甲基化水平越低,则克隆团囊胚率越高。研究结果对提高猪SCNT胚胎发育效率,改善现有的猪SCNT技术体系提供了基础。     

提高山羊卵丘细胞核移植效果的研究

 【目的】提高山羊卵丘细胞核移植效果。【方法】采用秋水仙胺提高山羊卵母细胞去核率,5-氮-2'-脱氧核苷（5-aza-dC）和曲古菌素A（TSA）影响山羊卵丘细胞核移植胚胎。【结果】0.5 μg?ml-1秋水仙胺处理山羊卵母细胞效果最好,胞质突起率达91.7%（P＜0.05）;通过比较盲吸去核法与化学辅助去核法的去核率及构建卵丘细胞核移植胚胎的体外发育能力发现,化学辅助去核法的去核率达100%,所构建的重构胚体外发育能力较高,桑椹胚率和囊胚率分别达25.2%和13.1%,显著高于盲吸去核法（P＜0.05）。采用0.01 μmol?L-1的5-aza-dC处 理山羊卵丘细胞,核移植胚胎桑椹胚率和囊胚率分别达30.4%和17.0%,显著高于其它各组（P＜0.05）;采用 400 nmol?L-1TSA处理山羊卵丘细胞,核移植胚胎桑椹胚率、囊胚率分别达31.5%和15.2%。【结论】化学辅助去核法的去核率显著高于盲吸去核法,采用0.01 μmol?L-1的5-aza-dC或400 nmol?L-1 TSA处理山羊卵丘细胞,效果最佳。     

Implications of static in vitro digestion of starch in the presence of dietary fiber

Interest in understanding the digestion behavior of starch in the presence of dietary fibers is growing due to the ability of dietary component to control the release and absorption of glucose. This presents an outstanding opportunity to improve the quality of food products by incorporating dietary fiber into starchy food products. The physicochemical properties of different fibers and their behavior in the gastrointestinal tract (GIT) differ. To test the efficacy of these different fibers on starch digestion, static in vitro digestion models under conditions that mimic the human GIT are frequently used. Indeed, many efforts have been committed to the development of various static in vitro protocols for starch digestion. Though not considered as the gold standard in digestibility studies in food science and technology, static simulated models provide a useful alternative to in vivo techniques for rapid screening of the digestibility of food products under conditions that simulate the human GIT. This review presents the current status and development of digestion techniques for simulating digestion conditions in the human GIT, with particular interest on starch digestion in the presence of dietary fiber in the three phases of digestions including the oral, gastric and the intestinal steps. This summary can benefit investigators in developing static in vitro digestion models designed to simulate starch digestion with relevant values of the quantifiable parameters, including pH, enzymes and simulated digestive fluids.     

牛卵母细胞显微操作机械去核方法的比较

分别采用挤压去核法、盲吸去核法和点击去核法对延边黄牛卵母细胞进行去核操作,在操作时间、成功率、去核率及重组胚后期发育等方面进行比较.结果表明,去核率上,盲吸法低于其他2种方法;去核成功率上,挤压法和点压法高于盲吸法;操作耗时上,挤压法和盲吸法耗时较短;3种方法所构建的重组胚卵裂率以及囊胚发育率均差异不显著.因此,挤压去核法是一种较适合延边黄牛卵母细胞去核的有效方法.     

广西巴马小型猪体细胞核移植体系的建立

通过建立广西巴马小型猪体细胞核移植培养体系,为后续转基因克隆猪生产及其相关研究提供最佳条件.试验探讨了盲吸法对猪卵母细胞的去核效率;利用分离到的广西巴马小型猪附睾成纤维细胞作供体构建重构胚胎,探讨6-二甲氨基嘌呤(6-DMAP)和细胞松弛素B(CB)对其后续胚胎发育的影响;利用分离到的广西巴马小型猪腹部成纤维作供体,探讨其对核移植胚胎的发育效率.结果表明:猪卵母细胞盲吸法去核的效率为73.75％;广西巴马小型猪附睾成纤维细胞作供体构建重构胚胎,6-DMAP和CB对其后续胚胎发育没有显著差异(分裂率和囊胚率分别为:89.89％and 6.74％vs.82.95％and 5.68％,P＞0.05);腹部成纤维细胞作供体,重构胚胎的融合率、分裂率和囊胚率分别为55.40％、67.53％和6.49％.采用盲吸法可以对体外成熟猪卵母细胞成功去核,广西巴马小型猪腹部成纤维细胞和附睾成纤维细胞均可作供体成功构建体细胞核移植胚胎,并可以发育到囊胚.     

Effects of in Vitro Maturation Time of Oocytes on Sheep Nuclear Transfer Efficiency

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).     

卵母细胞体外成熟时间对绵羊核移植效率的影响(英文)

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).     

卵母细胞体外成熟时间对绵羊核移植效率的影响(英文)

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).     

Factors affecting early embryonic development in cattle: relevance for bovine cloning

Female infertility represents a major challenge for improving the production efficiency in the dairy industry. Historically, fertility has declined whereas milk yield has increased tremendously due to intensive genetic selection. In vivo evidence reveals about 60% pregnancy loss takes place during the first month following fertilization. Meanwhile, early embryo development is significant for somatic cell nuclear transfer in cattle as a large proportion of cloned embryos fail to develop beyond peri-implantation stage. Oocyte quality is of utmost importance for the early embryo to develop to term for both fertilized and cloned embryos. Epigenetic reprogramming is a key process occurring after fertilization and critical roles of epigenetic modifiers during preimplantation development are now clear. Incomplete epigenetic reprogramming is believed to be a major limitation to cloning efficiency. Treatment of cloned embryos with epigenetic modifying drugs (e.g., Trichostatin A) could greatly improve cloning efficiency in both mice and cattle. Recently, the rapid progress in high-throughput sequencing technologies has enabled detailed deciphering of the molecular mechanisms underlying these events. The robust efficiency of genomic editing tools also presents an alternative approach to the functional annotation of genes critical to early development.     

G418处理核供体细胞对猪克隆胚胎体外发育效率的影响

【目的】探索G418处理供体细胞对其核移植胚胎发育效率的影响。【方法】利用G418单独处理猪成体成纤维细胞6 d后,收集细胞,利用荧光定量PCR的方法检测处理前后细胞中抗氧化应激、细胞凋亡相关基因的表达水平,运用亚硫酸盐结合测序法分别检测基因组重复序列LINE-1、微卫星的DNA甲基化状态,以及其核移植胚胎体外发育的能力。【结果】经不同质量浓度G418处理的供体细胞的抗氧化应激酶相关基因及细胞凋亡基因表达发生显著变化(P0.05),但其DNA甲基化水平没有发生改变(P0.05);经G418处理的供体细胞的核移植胚胎的体外发育效率显著低于对照组(P0.05)。【结论】G418处理供体细胞可能对其克隆胚胎体外发育效率有抑制作用。     

氧分压对猪卵母细胞成熟尧受精及早期胚胎发育的影响

研究利用两种氧分压（5%和20%）培养卵母细胞并进行IVF试验,同时使用两种氧分压培养孤雌（PA）胚胎、体外受精（IVF）胚胎和克隆(NT) 胚胎,以探寻猪的最佳体外培养环境。结果显示：低氧分压不利于猪卵母细胞的体外成熟,其成熟率极显著低于高氧培养的,但对低氧培养的卵母细胞进行IVF时,其卵裂率和囊胚率均极显著高于高氧培养的卵母细胞;IVF进行精卵孵育时,低氧环境下,其胚胎卵裂率和囊胚率均显著高于高氧环境下的;PA胚胎、IVF胚胎和NT胚胎用两种氧分压培养,低氧条件下IVF胚胎的发育能力高于高氧培养的,而PA胚胎和NT胚胎差异不显著。试验结果表明,低氧培养虽不利于卵母细胞的成熟,但有利于其以后的胚胎发育,且低氧环境下IVF胚胎的发育能力高于高氧环境下的。     

卵母细胞体外成熟时间对绵羊核移植效率的影响

[目的]为绵羊克隆试验中卵母细胞的体外成熟及去核时间提供参考。[方法]利用盲吸法结合荧光显微镜检查,对绵羊不同成熟时间卵母细胞去核的效率及其后续重构胚的发育进行对比。[结果]体外成熟培养19~21 h的绵羊卵母细胞在成熟率上显著高于体外成熟培养16~18 h(P(0.05),在去核成功率上显著高于体外成熟培养22~24 h(P(0.05);3个试验组在卵裂率、囊胚率上差异不显著(P(0.05),但是体外成熟培养19~21 h的试验组囊胚平均细胞数要显著高于其他2组(P(0.05)。[结论]体外成熟培养19~21 h的卵母细胞较适于作为受体细胞进行绵羊核移植,可以显著提高囊胚的质量。     

TSA浓度及处理时间对猪体细胞核移植胚胎体外发育影响

曲古抑菌素A(Trichostatin A,TSA)是一种组蛋白去乙酰化酶抑制剂.有研究表明TSA处理可以提高核移植胚胎的发育率.为探讨TSA对猪核移植胚胎发育的作用,试验重点研究了TSA浓度及处理时间对核移植胚胎体外发育的影响,同时也探索了TSA对不同供核细胞构建的重构胚体外发育的影响.结果表明,40 nmol·L-...     

胚胎移植方法和受体母猪因素对克隆猪生产效率的影响

【目的】建立高效的胚胎移植技术体系,以提高克隆猪的生产效率。【方法】比较不同移植方法和不同受体母猪状况的胚胎移植分娩率和克隆猪的出生效率,利用体外发育能力相似的不同品种和发育阶段的克隆胚胎混合移植确定最佳的受体母猪发情同期时间。【结果】克隆胚胎经由输卵管伞移植比输卵管打孔移植具有更高的移植分娩率和克隆猪效率（2.2% 和0.4%,P＜0.01）,而胚胎移植后辅助人工授精会降低克隆效率（0.6% 和 2.2%,P＜0.01）。利用经产母猪作为移植受体比青年猪受体具有更高的克隆效率（3.0%和0.8%,P＜0.01）和平均窝产仔数（（5.5±0.7）头和（2.7±0.3）头,P＜0.05）。受体母猪发情时间比胚胎激活时间晚12-36 h组的克隆效率分别为显著（P＜0.05）和极显著（P＜0.01）,高于晚0 h和早12-24 h 组的克隆效率（2.0%、0.5%和0%）。最佳的受体母猪发情时间为晚于克隆胚胎激活后24 h,克隆效率达3.0%。【结论】选择自然发情时间晚于克隆胚激活后24 h的经产母猪为受体,通过输卵管伞端移植胚胎,能够获得较高的克隆效率,是猪克隆胚胎移植的较理想方法。     

小型猪体细胞克隆试验

[目的]建立体细胞克隆小型猪的技术平台并了解克隆效率。[方法]运用体细胞核移植技术,以小型猪胎儿成纤维细胞作为核移植供体细胞,以体外成熟卵母细胞作为核移植受体细胞进行体细胞克隆猪生产,并利用STR序列对所出生的克隆猪进行个体识别。[结果]将重构胚胎移植到5头代孕母猪体内,其中一头代孕母猪成功产下2头克隆小型猪。经过个体识别试验证实克隆小型猪来自供核细胞,与代孕母猪无直接亲缘关系。[结论]克隆小型猪的成功出生表明所建立的技术平台适于小型猪的克隆。     

杂草致病菌株GD-0221的分离、鉴定与除草潜力

为明确杂草致病菌株GD-0221的除草活性与作物安全性,利用组织分离法从患病甘草(Glycyrrhiza uralensis)根部分离获得杂草致病菌株GD-0221,通过形态学和分子生物学相结合确定该菌株的分类地位,利用菌饼接种法研究该菌株对5种阔叶杂草离体叶片的致病性,用发酵液喷雾接种法研究其对5种阔叶杂草的盆栽致病性和对青海主栽作物的安全性。根据菌株形态和DNA序列分析结果,将该菌株鉴定为尖孢镰刀菌(Fusarium oxysporum)。该菌株对5种阔叶杂草密花香薷(Elsholtzia densa)、藜(Chenopodium album)、冬葵(Malva verticillata)、酸模叶蓼(Polygonum lapathifolium)、萹蓄(Polygonum aviculare)均具有致病性,活体接种试验中杂草发病率为33.33%~100.00%,对密花香薷的致病效果最突出。作物安全性方面,该菌株对青稞(Hordeum vulgare)、小麦(Triticum aestivum)表现安全,而对油菜(Brassica campestr)、蚕豆(Vicia faba)和豌豆(Pisum sativum)表现不安全。GD-0221菌株具有开发成为禾本科作物田防除阔叶杂草微生物除草剂的潜力。     

Effects of red and blue LEDs on in vitro growth and microtuberization of potato single-node cuttings

The objectives of this study were to investigate the effects of red and blue LEDs on in vitro growth and microtuberization of potato (Solanum tuberosum) single-node cuttings. Explants were incubated under 6 light treatments: 100% red LEDs (R), 75% red LEDs+ 25% blue LEDs (3RB), 50% red LEDs+ 50% blue LEDs (RB), 25% red LEDs+ 75% blue LEDs (R3B), 100% blue LEDs (B) and white LEDs (W). Most of the growth and physiological parameters were significantly higher in 3RB than W. Enhancement of leaf area and chlorophyll concentrations were obtained in B. Leaf stomata were elliptical with the lowest density in 3RB. However, those in W were round in shape, and those with the smallest size and the highest density were observed in R. Most of the characteristics of microtuberization were also improved in 3RB. The combined spectra of red and blue LEDs increased the number of large microtubers. The fresh weight of individual microtubers in R and W were increased, but not their number. These results suggest that, of the treatments assessed, 3RB is optimal for the in vitro growth of potato plantlets and the combination of red and blue LEDs is beneficial for microtuberization.     

基于粗提物的水稻条纹病毒体外“抓帽”体系

【背景】 布尼亚病毒目（Bunyavirales）和正黏病毒科（Orthomyxoviridae）病毒从帽子结构下游10—20个碱基处切割寄主mRNA,以5′端切割产物（帽子序列）作为引物起始自身基因组的转录,生成含一段异源帽子序列的病毒mRNA,这一过程称为“抓帽”。水稻条纹病毒（rice stripe tenuivirus, RSV）是一种布尼亚病毒,其“抓帽”机制还不甚清楚。【目的】 研究RSV粗提物能否从溶液中的外源mRNA“抓帽”,以建立一种便捷的体外体系,用于解析RSV“抓帽”和转录机制。【方法】 以PEG沉淀和超速离心法粗提RSV,利用聚丙烯酰胺凝胶电泳（SDS-PAGE）和质谱技术分析粗提物成分。然后取少量粗提液,加入富含珠蛋白（globin）mRNA的兔网织红细胞裂解液（rabbit reticulocyte lysate,RCL）和合适浓度的金属离子配制体外“抓帽”体系。待体外反应结束,提取体系中总RNA,以巢式RT-PCR扩增含珠蛋白-α mRNA帽子序列的RSV mRNA,并对其进行克隆,通过序列分析比较RSV体外“抓帽”与体内“抓帽”的异同。【结果】从100 g感染RSV的水稻叶片获得RSV粗提液2 mL。除RSV病毒粒子外,粗提液中还含核酮糖-1,5-二磷酸羧化酶等多种叶绿体蛋白。取2 μL粗提液,加入4 mmol·L^-1 MgCl₂、2 mmol·L^-1 NTP、0.8 U·μL^-1 RNA酶抑制剂和8 μL RCL,配成20 μL反应体系,30℃孵育1.5 h后,巢式RT-PCR扩增到了目的条带,表明RSV粗提物能切割溶液中的珠蛋白-α mRNA,并利用其帽子序列合成自身mRNA。在反应体系中加入帽子结构类似物m⁷G（5′）ppp（5′）G后进行相同反应,目的条带变淡,且变淡程度与所加入m⁷G（5′）ppp（5′）G的浓度成正比,表明识别帽子结构是RSV从溶液中切割利用珠蛋白-α mRNA的前提。对巢式RT-PCR产物克隆和测序后分析发现,与在体内的情况类似,RSV从帽子结构下游的A或C处切割珠蛋白-α mRNA,得到的帽子序列与病毒模板链3′端的U或G配对后引发转录。在利用珠蛋白-α mRNA帽子序列进行转录的过程中,RSV频繁使用引发与重配,且在合成核蛋白基因NP时比在合成主要非核蛋白基因NCP时使用该机制的频率更高。【结论】 RSV粗提物能从溶液中的mRNA“抓帽”,且在切取和利用帽子序列的方式上,其表现与细胞内的RSV无明显差别。因而,粗提物可以用来解析RSV的“抓帽”机制,甚至用于研究RSV的转录。