体细胞克隆太湖猪出生

以中国太湖猪胎儿成纤维细胞为核供体,以体外成熟卵母细胞为核受体,以杜洛克猪为代孕母猪,进行了太湖猪体细胞克隆技术的研究,成功地获得了首例体细胞克隆太湖猪仔猪.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.本研究将为体细胞克隆技术在我国太湖猪育种、建立人类疾病模型等研究方面提供可行的技术方法.     

我国首例成体体细胞作为核供体的东北民猪克隆成功

10月12日，在哈尔滨市三元畜产实业有限公司种猪场，由东北农业大学刘忠华博士带领的《东北民猪体细胞核移植研究》课题组与哈尔滨市三元畜产实业有限公司合作，培育出的我国首例采用成体体细胞作为核供体的3头健康雌性克隆东北民猪以剖腹产的方式顺利出生．克隆猪各项形态特征与供核体细胞个体完全一致。     

东北民猪被成功克隆

我国首例采用成体体细胞作为核供体的克隆东北民猪在哈尔滨诞生。这是我国第二例取得成功的克隆猪。     

东北民猪被成功克隆

东北农业大学、哈尔滨科技局及合作企业合作研究的采用成体体细胞作为核供体克隆猪技术取得成功。采用此技术克隆出来的3头东北民猪，自出生以来发育正常，长势良好。     

靶向O型口蹄疫病毒shRNA转基因猪体细胞抗病毒活性研究

为研究靶向O型口蹄疫病毒(FMDV)VP1基因shRNA转基因猪体细胞的抗病毒活性,本实验在成功培育转基因克隆猪的基础上,通过分离与培养转基因猪体细胞,对其shRNA进行PCR和Southern blot检测,并将FMDV感染体细胞中,通过细胞病变(CPE)、间接免疫荧光试验(IFA)和实时荧光定量PCR(Real-time PCR)分析转基因猪体细胞抗FMDV活性。结果表明,转基因猪体细胞基因组DNA中携带有靶向FMDV VP1基因的shRNA基因片段。与非转基因猪体细胞相比,接种FMDV转基因猪体细胞其出现CPE的时间延迟,细胞内病毒含量显著降低,细胞感染病毒36 h时,对细胞中FMDV VP1基因抑制效率为53.6%。表明该靶向FMDV shRNA转基因克隆猪体细胞在体外具有良好的抗病毒活性。本研究为进一步在体内评价转基因动物的抗病毒活性奠定了基础。     

电融合/激活对于巴马小型猪体细胞克隆胚胎生产的影响

本试验旨在建立适用于猪体细胞克隆的融合/激活体系(试验1),优化融合/激活条件以提高重构卵融合率和激活率,为生产足够数量的克隆胚胎用于胚胎移植最终诞生体细胞克隆猪奠定条件。首先比较脉冲时间确定适合体细胞克隆的最佳融合/激活参数,然后利用此参数验证巴马小型猪耳部成纤维细胞克隆胚胎的发育潜能。结果显示:场强100 V/mm,3次电脉冲,脉冲时间为30μs时适于猪体细胞克隆。在试验2中,利用巴马小型猪耳部成纤维细胞为供体细胞构建体细胞克隆胚胎,得到了较高的卵裂率(83.1%)和囊胚发育率(12.4%)。     

巴马小型猪主要细胞因子的克隆及序列分析

为了丰富广西巴马小型猪细胞因子生物学数据,本试验采取广西巴马小型猪的外周血,分离淋巴细胞,提取总RNA,利用设计的引物进行RT-PCR扩增,将克隆出的与目的基因大小相符的片段回收,再与T载体连接,转化到大肠杆菌DH5α中,筛选阳性克隆,将重组菌测序后提交NCBI,比较克隆序列与已知序列的同源性,并利用软件对克隆序列进行分析。结果表明,已克隆出的巴马小型猪细胞因子IL-2、IL-12、IFN-γ、TNF-α基因序列长度分别为338、377、380、402 bp,测序结果均与目的基因预期估计值吻合。序列分析结果发现与已知的家猪或野猪的相应基因同源性较高,IL-2、IL-12、IFN-γ、TNF-α序列同源性分别为98.9%、99.5%、99.2%、100%。研究巴马小型猪细胞因子基因序列不仅丰富了生物学数据,而且为细胞因子的功能研究提供了依据,也为细胞因子的定量检测奠定了基础。     

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。     

我国第1头体细胞克隆猪诞生

中国农业大学成功地获得我国第1头体细胞克隆借,这是我国独立自主完成的首例体细胞克隆猪,填补了我国在这一领域的空白。     

鸡舍纵向通风系统主要性能指标的检查方法

纵向通风系统是标准化鸡舍内环境调控的必需设施,该系统除鸡舍的外围护结构外,风机、湿帘和挡风板则是最重要的通风设备。为使鸡舍保持良好的内部空气质量和适宜的温湿度环境,鸡舍启用前后,应对该系统所有的设备进行认真的检查维修,消除潜在的隐患,减少因通风不良而导致的饲养损失。     

Reproductive performance and expression of imprinted genes in somatic cell cloned boars

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin‐like growth factor ‐2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot‐and‐mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.     

克隆种公猪生长状况和繁殖性能评价

体细胞核移植技术可以成功用于克隆性能优良的种用家畜,理论上克隆个体与供体在遗传上是完全相同的。为验证该理论,本试验检测了克隆种公猪自身在出生后的生长发育指标以及克隆种公猪与供核种公猪精液品质各项指标、情期受孕率、窝产仔数、产活仔数、断奶活仔数、仔猪初生重、仔猪断奶重和平均日增重等指标,探讨了克隆种公猪与供核种公猪在繁殖性能以及遗传性能之间的关系。结果显示,克隆种公猪与供核种公猪在各检测指标之间均不存在显著差异(P>0.05),提示克隆种公猪与供核种公猪在繁殖性能和遗传性能上无显著差异,可以作为种公猪用于扩繁。     

Assessment of reproduction and growth performance of offspring derived from somatic cell cloned pigs

Since cloned pig was successfully produced, a new opportunity for porcine breeding industry to conserve genetic resources has been opened. However, there has been no report to investigate whether both somatic cell nuclear transfer (SCNT) pigs and their offspring have the characteristics of the donor breed. In this study, we compared the reproductive and growth performance of American Large White boars cloned by SCNT with the donor boar, and analyzed the test parameters, including semen quality, re‐service rate, rate of parturition, and average daily gain. The results showed that these cloned boars and the donor boar had no significant differences in the tests (P > 0.05) and the growth performance of their offspring was similar to the naturally bred American Large White pigs. In summary, the reproductive and growth performance of cloned pigs are similar to the donor pig and within the normal range. This suggests that pigs cloned by SCNT have the potential to be used in reproduction and breeding.     

Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan

Since the first somatic cell cloned calves were born in Japan in 1998, more than 500 cloned cattle have been produced by somatic cell nuclear transfer and many studies concerning cloned cattle and their offspring have been conducted in this country. However, most of the results have been published in Japanese; thus, the data produced in this country is not well utilized by researchers throughout the world. This article reviews the 65 reports produced by Japanese researchers (62 written in Japanese and 3 written in English), which employed 171 clones and 32 offspring, and categorizes them according to the following 7 categories: (1) genetic similarities and muzzle prints, (2) hematology and clinical chemistry findings, (3) pathology, (4) growth performance, (5) reproductive performance, (6) meat production performance and (7) milk production performance. No remarkable differences in health status or reproductive performance were found among conventionally bred cattle, somatic cell cloned cattle surviving to adulthood and offspring of somatic cell cloned cattle. Similarities in growth performance and meat quality were observed between nuclear donor cattle and their clones. The growth curves of the offspring resembled those of their full siblings.     

体细胞核移植生产转fat-1基因克隆Holstein牛

为培育含ω-3多聚不饱和脂肪酸(ω-3polyunsaturated fatty acids,PUFAs)丰富的转基因奶牛新材料,本试验将fat-1基因转染到Holstein奶牛胎儿成纤维细胞,筛选获得的转基因阳性克隆细胞株,通过核移植方法构建转基因重构胚胎,比较了非转基因与转基因重构胚体外发育情况,并移植到代孕母牛子宫角。妊娠足月产下犊牛后,对犊牛进行转基因的DNA以及mRNA的表达鉴定。结果显示,非转基因与转基因重构胚囊胚率分别为32.1%和28.1%,两者之间无显著差异(P0.05)。受体母牛的妊娠率为66.7%(6/9),其中3头母牛妊娠足月(50%)。自然分娩3头克隆牛,体细胞克隆牛的效率为20%(出生小牛头数/移植胚胎数)。经PCR鉴定,这3头小牛中有1头为转fat-1基因阳性,其余2头均为阴性,转基因阳性率为33.3%。     

Reproductive and growth performance in Jin Hua pigs cloned from somatic cell nuclei and the meat quality of their offspring

Somatic cell cloning is expected to be a valuable method for conserving genetic resources in pigs. In this study, we compared the reproductive and growth performance of Jin Hua cloned pigs with that of naturally bred Jin Hua pigs. In addition, we generated offspring from the cloned sows and examined the productivity and quality of meat in the progeny. The birth weights and growth rates of somatic cell-cloned pigs were similar to those of Jin Hua pigs. The cloned pigs reached puberty very early, and this is typical of the Jin Hua breed. Furthermore, reproductive performance, in terms of traits such as gestation period, litter size, and raising rate in the cloned pigs were similar to Jin Hua pigs. Although the offspring of the cloned (OC) pigs had lower birth weights than the Jin Hua breed, the daily weight gain of the OC pigs was significantly higher, especially at the finishing stage. The carcass quality of the OC pigs had similar characteristics to the Jin Hua breed, namely thick back fat and a small loin area. Furthermore, the meat qualities of the OC pigs were similar to those of Jin Hua pigs in terms of intramuscular fat content and tenderness. These results demonstrate that cloned pigs and their offspring were similar to the Jin Hua breed in most of the growth, reproductive, and meat productive performances. This strongly suggests that pigs cloned from somatic cell nuclei have the potential to be a valuable genetic resource for breeding.     

H19和IGF2R基因在体细胞克隆猪各组织中的甲基化状态

为了探求新生克隆猪可能的死亡原因以及是否存在不完全的DNA甲基化重编程,本试验运用亚硫酸氢盐测序法分别检测了H19基因和IGF2R基因差异甲基化区（DMR）在新生死亡克隆猪和同期正常猪心脏、肝脏、脾脏、肺脏和肾脏中的甲基化状态。结果发现,H19基因DMR在克隆猪肺脏中表现为超甲基化,极显著高于正常猪（95．20％VS46．80％P〈0．01）,且10个测序克隆中存在2处连续的全甲基化CpG位点（4-9位、12-S17位）,而在其他组织中甲基化差异不显著（P〉0．05）;IGF2R基因DMR在肝脏中处于超甲基化状态,显著高于正常猪（80．00％V839．41％P〈0．05）,而在肺脏中为去甲基化状态,板显著低于正常猪（14．71％VS66．47％P〈0．01）,在其他组织差异不显著（P〉0．05）。结果说明,在死亡克隆猪中,H19基因DMR在肺脏和IGF2R基因在肝脏与肺脏中存在不完全的DNA甲基化重编程,这可能是导致克隆动物死亡的因素之一。     

应用体细胞克隆技术和胚胎移植技术生产克隆水牛

为了解决克隆水牛技术在供核细胞、受体细胞中的选用问题,探索不同条件对移植受孕的影响,完善克隆技术的理论体系。试验以摩拉奶水牛的耳成纤维细胞作为供核细胞,本地水牛卵母细胞为受体胞质进行细胞克隆构建重组胚胎;将发育5～10 d的重组胚胎移植于本地水牛、杂交水牛的子宫内,观察不同发情方式、胚胎类型、胚龄及季节对移植受孕的影响。结果表明:水牛的克隆胚胎妊娠率较低,分别为12.05%、12.04%、13.95%。自然发情和同期发情对克隆胚胎移植受胎率的影响差异不显著;鲜胚胎移植186头,受胎22头,受胎率为11.83%(22/186);冻胚胎移植174头,受胎23头,受胎率为13.22%(23/174)。克隆水牛从出生至12月龄体尺增长明显高于本地水牛;水牛克隆胚胎移植受胎在秋季最好,秋季受胎数占全年的53.33%,显著高于其他季节。