小鼠-猪种间体细胞克隆试验

用3代~5代小鼠胎儿成纤维细胞(MEF)作核供体细胞,体外成熟培养44 h~46 h、排出第1极体(PbI)的猪卵母细胞去核后,进行小鼠-猪种间核移植操作。Hoechst 33342染色检查去核效果,去核率为84.2%(27/32)。共培养68枚小鼠-猪种间核移植重构胚,其中48枚发生卵裂,卵裂率为70.6%,13枚胚胎发育至8-细胞,发育率达27.1%(13/48)。     

体细胞核移植生产梅山猪

本研究以中国优良地方品种梅山猪为材料,采用胰酶消化法获得猪胎儿成纤维细胞,通过使用Y染色体SRY基因引物SRY-1、SRY-2进行PCR扩增,结果发现,雄性胎儿样本能扩增出250 bp的Y染色体特异性基因片段,而雌性胎儿样本未扩增出此条带;G1、G2、G3、G4和G5代的融合效率差异不显著(P>0.05),但G5代作为供体细胞核移植的重组胚胎卵裂率显著高于G1、G2、G3和G4代(P<0.05);使用G5代的胎儿成纤维细胞作为供核细胞,通过手术移植的方法,将重构胚胎移植到二元后备母猪体内,结果成功地获得了体细胞克隆梅山仔猪,并且仔猪全部为雄性胎儿,说明该性别鉴定方法不但操作简单,而且准确度高;经微卫星DNA 多态性鉴定,确定克隆猪来自供核细胞,与代孕母猪无亲缘关系。本研究将为中国优质猪品种改良、保种、性别控制及建立人类疾病模型等研究提供有效可行的方法,为批量生产克隆优秀种猪提供了坚实的理论基础。     

供体细胞对猪体细胞克隆胚胎早期发育的影响

以中国农业大学实验用小型猪香猪胎儿成纤维细胞、成年耳成纤维细胞和颗粒细胞3种细胞系为供体细胞进行核移植。比较了血清饥饿法和接触抑制法处理胎儿成纤维细胞诱导进入G0／G1期的效率，发现二者差异不显著（P〉0．05），血清饥饿2d和4d差异不明显，同样接触抑制2d和4d差异也不显著（P〉0．05）。系统研究了影响克隆胚胎发育的供体因素：血清饥饿与否、细胞形态、细胞类型及个体差异等，结果表明：血清饥饿处理对克隆胚的早期发育没有明显的促进作用；圆形光滑细胞有利于细胞融合，对早期发育无显著影响（P〉0．05）；不同个体、不同类型的供体细胞对克隆胚囊胚发育率有一定的影响。     

体细胞克隆小型猪主要性能指标的测定

通过与普通巴马小型猪的生长发育规律、繁殖性能、生理生化指标和屠宰肉质性能等的比较,了解小型克隆猪的主要性能指标,以期为克隆猪安全性评估及其产业化健康发展提供借鉴.结果表明,体细胞克隆巴马小型猪与普通猪的各项性能指标差异不显著.这种相似性在一定程度上说明了体细胞克隆猪的产业健康性和安全性.     

我国第一例成体体细胞克隆猪出生

据东北农业大学消息,我国第一例采用成体体细胞作为供体的3只克隆东北民猪10月12日在黑龙江省哈尔滨市出生.此举标志我国在克隆猪领域的研究工作达到国际先进水平.     

猪颗粒细胞和胎儿成纤维细胞的分离培养与冷冻保存试验报告

本试验利用从成熟后的猪卵母细胞中获得的颗粒细胞与胎儿成纤维细胞作为下一步猪体细胞核移植的供核细胞作好准备,为转基因猪的研究奠定了基础。对从成熟卵母细胞中获得的颗粒细胞进行分离培养和冷冻保存;采用室温消化法分离培养猪胎儿成纤维细胞。结果表明,接种后的原代颗粒细胞和猪胎儿成纤维细胞经过5～6d后均可连生铺满皿底,细胞排列有序,可用于继代培养;猪胎儿成纤维细胞进行冷冻解冻后可以存活。利用成熟后的猪卵母细胞的颗粒细胞,可以简单而快速地分离与培养;通过室温消化法可以分离培养猪胎儿成纤维细胞,并且经过冷冻解冻后可以复苏存活,其存活率为80.2%。     

中国第1例成体体细胞克隆猪出生

据东北农业大学消息,我国第1例采用成体体细胞作为供体的3只克隆东北民猪于2006年10月12日在哈尔滨市三元畜产实业有限公司种猪场出生。表明我国在克隆猪领域的研究工作达到国际先进水平。     

我国关于太湖猪与西方猪的杂交效果

对我国太湖猪与西方猪的杂交效果进行了综述。结果表明：纯种太湖猪与纯种西方相比,其产仔性能较高,但能性能,胴体品质,实生个体重和４５日龄个体重都不及纯种西方猪的好;西方猪可用来提高中国太湖猪的肥育性能和胴体品质,但杂种猪的产仔性能较纯种太湖猪有所下降;     

太湖猪二元杂交效果的试验研究

利用太湖猪与引进的瘦肉型猪种进行二元、三元杂交,具有明显的经济效益,为国内外学者所注目。C.legault和J.C.Caritez(法国)概括太湖猪有五大特点,即性成熟早;繁殖力高;母性强而全面;省饲料;肉味鲜美。实践证明确实如此。现仅就二花脸系和梅山系太湖猪二元杂交结果报道如下。一、材料与方法 (一)供试猪种由江苏省引进品种特征明显,体形外貌典型的二花脸猪26头(母20头,公6头),梅山猪10头,形成两个太湖纯系繁殖群。     

水牛体细胞连续核移植的效果

为探讨水牛体细胞连续核移植的效果,以水牛胎儿成纤维细胞为核供体,进行了水牛体细胞连续核移植。结果显示,连续核移植的融合率显著高于原代核移植(87.9%vs76.2%,P<0.05),但两者之间的分裂率和囊胚率没有显著差异(P>0.05);这说明水牛体细胞核移植胚胎可被再次克隆而不降低其发育能力,水牛体细胞连续核移植是可行的。     

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.     

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.     

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。     

Propagation of elite rescue dogs by somatic cell nuclear transfer

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.     

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.     

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer

BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos.     

单胚胎RT-qPCR对猪体细胞核移植囊胚全能性的分析

为了检测猪体细胞核移植囊胚的质量和全能性基因表达量,采用体细胞核移植技术制备克隆胚胎并于体外培养5 d后获得囊胚,对照组为从人工授精5 d后的长白母猪体内获取的体内囊胚。在高倍镜下检测两组囊胚的形态,用Hoechest 33342染色细胞核DNA,记录囊胚细胞总数;建立单胚胎cDNA的制备方法,并用qPCR检测囊胚中全能性基因(Oct4、Nanog、Sox2)的表达量。结果表明,与体内囊胚相比,猪体细胞核移植囊胚质量较差,细胞数目显著降低(分别为110±10.3和54±12.6);并且全能性基因表达量显著下降(P<0.05)。由此可见,全能性基因表达量偏低是影响猪体细胞核移植囊胚发育能力的因素之一。