Preparation and Properties of Aqueous SCNTs Dispersion based on A UV-curable Polymeric Dispersant

A novel photosensitive copolymer P（SS-co-AA-g-GMA）（PSAG） was synthesized and utilized to noncovalently functionalize pristine single-walled carbon nanotubes（SCNTs）. PSAG was highly effective for the solubilization of SCNTs in water and validated by UV-vis absorption spectra experiments, resulting in homogeneous and stable PSAG-SCNT aqueous dispersion. The microstructure of SCNTs was observed through Raman spectroscopy and transmission electron microscopy. In addition, compared with the two common polymeric dispersants of Triton X-100 and PSS, PSAG demonstrated more effective performances for dispersing SCNTs under identical conditions. Furthermore, the photosensitive PSAG-SCNTs can be crosslinked after UV irradiation, leading to significant improvement in the water resistance of SCNT films. UV-cured films can be transferred to plastic wrap to form a flexible film with high electrical conductivity.

体细胞核移植技术及应用

体细胞核移植(somatic cell nuclear transfer,SCNT)是一种能使已分化的体细胞获得全能性(totipotency)的技术,在发育生物学、生物医学研究和农业应用方面具有重要价值。该技术主要包括卵细胞去核和核供体细胞植入两个环节。文章综述了SCNT技术的原理、流程、应用等,以期为SCNT技术的研究与应用提供参考依据。

Computational Analysis of Heat and Mass Transfer in Magnetized Darcy-Forchheimer Hybrid Nanofluid Flow with Porous Medium and Slip Effects

A computational analysis of magnetized hybrid Darcy-Forchheimer nanofluid flow across a flat surface is presented in this work.For the study of heat and mass transfer aspects viscous dissipation,activation energy,Joule heating,thermal radiation,and heat generation effects are considered.The suspension of nanoparticles singlewalled carbon nanotubes(SWCNTs)and multi-walled carbon nanotubes(MWCNTs)are created by hybrid nanofluids.However,single-walled carbon nanotubes(SWCNTs)produce nanofluids,with water acting as conventional fluid,respectively.Nonlinear partial differential equations(PDEs)that describe the ultimate flow are converted to nonlinear ordinary differential equations(ODEs)using appropriate similarity transformation.The ODEs are dealt with numerically by means of MATLAB’s inbuilt routine function bvp4c.Velocity,temperature,and concentration profiles are explained pictorially whereas Sherwood number,local skin friction coefficient,and Nusselt number values are represented through bar charts.Thermal radiation and activation parameters shows direct impact on flow field.Furthermore,hybrid nanofluid admits a higher magnitude of velocity and temperature than nanofluid,but the concentration profile exhibits the opposite trend.The notable findings of the present investigation have significant applications in heat combustion and cooling chambers,space technology,the ceramics industry,paint and conductive coatings,bio-sensors,and many more.

猪成纤维细胞细胞周期同期化对核移植胚胎发育的影响

供体细胞所处的细胞周期及细胞周期同期化的方法对于体细胞核移植（somatic cell nuclear transfer，SCNT）的成功非常重要，本研究对血清饥饿培养处理与培养至完全汇合后的猪成纤维细胞周期同期化水平进行了检测。利用不同方法对猪成纤维细胞同期化处理后，通过流式细胞仪对细胞的细胞周期分布比率进行了检测。将细胞进行血清饥饿24～72h，显著地增加了Go／G。期的细胞百分率（92．2％-93．7％VS．77．8％，P〈0．05）。将细胞培养至完全汇合后再培养24-48h，GgG。期的细胞比例类似于血清饥饿法（94．4%，89．6%）。血清饥饿24h后，置换为10％FBS能逆转至生长期。用这两种不同方法处理后的体细胞作为核移植的供体构建重构胚，分裂率与囊胚率差异不显著（P〉0．05）。结果表明，猪成纤维细胞通过血清饥饿法或者培养至汇合完全均能有效地将细胞周期同期化至G0/G1期，且均可作为体细胞核移植的供体细胞。

1例死亡克隆牛主要脏器组织学观察及胎盘印记基因甲基化水平分析

虽然体细胞克隆技术已经在多种动物获得成功,但是仍然存在效率低、克隆胚胎发育异常等问题,严重限制了该技术的应用。为提高克隆效率,探讨克隆动物发育异常的原因,本研究对1例死亡克隆牛主要内脏器官进行了组织学观察,并对其胎盘印记基因甲基化状态进行分析。结果表明,此例死亡克隆牛主要内脏器官除肾脏外,均不同程度出现充血、增生及淋巴细胞浸润等病理变化。胎盘增生明显并伴有充血,胎盘中印记基因H19及Peg-10的甲基化水平分别为84.6%和88.6%,显著高于50%,出现超甲基化状态。推测体细胞核移植过程影响了供核细胞的表观重编程,导致了印记基因H19及Peg-10的迷乱的DNA甲基化状态,并影响胎盘及内脏器官的正常发育。

外源激素组合控制青春期前奶山羊卵泡超数同步发育及其卵母细胞发育潜能的评价(英文)

本研究的目的是探索自青春期前奶山羊获取大量可用于体细胞核移植的卵母细胞的可能性。为此,本研究比较了几种不同组合的激素处理方法(对照、FSH、E2-P4和E2-P4-FSH)对出生39-60日龄的奶山羊卵巢大小、卵泡数量和卵泡大小的影响:同时将出生39-120日龄奶山羊按年龄分成三组来研究年龄对激素处理时招募起始生长卵泡数量的影响:然后,比较了来自E2-P4- FSH和FSH处理的早青春期前奶山羊卵巢上直径大于3mm卵泡中卵母细胞减数分裂能力;最后,通过SCNT方法验证E2-P4-FSH处理的早青春期前奶山羊卵巢上直径大于3mm卵泡中卵母细胞的发育能力。在四组激素处理的早青春期前奶山羊中,E2-P4-FSH处理组的卵巢最大、卵泡(直径大于3 mm)数量最多。在不同的年龄组中,39-60天组奶山羊卵巢上直径大于3mm的卵泡数量显著多于61-90天和91-120天组的。卵母细胞减数分裂能力的分析结果表明,来自E2-P4-FSH处理组的卵母细胞减数分裂能力显著高于FSH处理组的卵母细胞。与E2-P4-FSH处理后的成年奶山羊卵母细胞相比,早青春期前奶山羊卵母细胞发育能力较低:卵母细胞成熟后,作为受体用于体细胞核移植后的克隆囊胚发育率低于成年奶山羊(15.3%versus 22.1%,P<0.01)。然而,早青春期前的奶山羊经E2-P4-FSH处理后,自每头羊卵巢上直径大于3mm的卵泡数显著高于成年奶山羊(108±10.3 versus 28±5.0),因此,每头早青春期前奶山羊产生的克隆囊胚绝对数量显著高于成年奶山羊(7.1±2.7 versus 4.2±1.4)。由此,从本研究可以得出结论:E2-P4-FSH处理的早青春期前奶山羊能够为体细胞核移植研究提供相对多数量的具备一定发育能力的成熟卵。

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。

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.

Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis

Somatic cell nuclear transfer （SCNT） and parthenogenesis are alternative forms of reproduction and development, building new life cycles on differentiated somatic cell nuclei and duplicated maternal chromatin, respectively. In the preceding paper （Sun F, et al., Cell Res 2007; 17：117-134.）, we showed that an ＂erase-and-rebuild＂ strategy is used in normal development to transform the maternal gene expression profile to a zygotic one. Here, we investigate if the same strategy also applies to SCNT and parthenogenesis. The relationship between chromatin and chromatin factors （CFs） during SCNT and parthenogenesis was examined using immunochemical and GFP-fusion protein assays. Results from these studies indicated that soon after nuclear transfer, a majority of CFs dissociated from somatic nuclei and were redistributed to the cytoplasm of the egg. The erasure process in oogenesis is recaptured during the initial phase in SCNT. Most CFs entered pseudo-pronuclei shortly after their formation. In parthenogenesis, all parthenogenotes underwent normal oogenesis, and thus had removed most CFs from chromosomes before the initiation of development. The CFs were subsequently re-associated with female pronuclei in time and sequence similar to that in fertilized embryos. Based on these data, we conclude that the ＂erase-and-rebuild＂ process observed in normal development also occurs in SCNT and in parthenogenesis, albeit in altered fashions. The process is responsible for transcription reprogramming in these procedures. The ＂erase＂ process in SCNT is compressed and the efficiency is compromised, which likely contribute to the developmental defects often observed in nuclear transfer （nt） embryos. Furthermore, results from this study indicated that the cytoplasm of an egg contains most, if not all, essential components for assembling the zygotic program and can assemble them onto appropriate diploid chromatin of distinct origins.

Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

曲古抑菌素A在体细胞核移植中的应用

体细胞核移植（SCNT）技术已成功获得多种哺乳动物的克隆后代，但一直以来克隆的成功率都很低，并且存活个体大多表型异常。研究表明，重构胚的重编程不完全是导致克隆动物存活率低下和发育异常的主要原因之一。DNA甲基化、组蛋白乙酰化都是影响重编程的重要因素，曲古抑菌素A（TSA）是链霉素的代谢产物，同时也是一种组蛋白去乙酰化酶抑制剂，能降低DNA甲基化水平，促进重编程率，进而提高克隆效率。本文对TSA的生物学功能及其在动物SCNT中的应用进行了探究。

培养液添加物与去核方法对猪体细胞核移植效率的影响

综述了在卵母细胞的成熟、去核和后期发育过程中添加化学成分对体细胞核移植效率的影响,以期对改进体细胞核移植效率提供参考。

体细胞克隆猪皮肤和毛发色素脱失的研究

利用杜洛克猪供体细胞已成功培育了9头克隆猪,其中1头在初情期皮肤和毛发出现明显的色素脱失,本试验对该头克隆猪的体细胞核移植异常的病因进行了研究。假设Waardenburg综合征的相关基因(Mitf、Pax-3、Sox-10、Slug和Kit)与克隆猪体细胞核移植(scNT)所致的色素脱失密切相关。试验从受scNT影响和未受scNT影响的猪耳组织中分别提取总RNA,运用反转录聚合酶链反应扩增Mitf、Pax-3、Sox-10、Slug的转录体和Kit基因,测序并分析。cD-NA序列反转录结果表明,毛发逐渐褪色的scNT猪的这些基因并没有发生突变。尽管没有发现任何突变,但与未受scNT影响的猪相比,Waardenburg氏综合征相关的这些基因在受scNT影响猪中的表达出现明显的负调控。基因表达的负调控可能产生一种黑素细胞不稳定的新表型,并最终导致色素沉着逐渐消失。

克隆伦理学

克隆细胞的基因组是母细胞或“祖”细胞几乎完全相同的副本，基因组克隆有两种方法——分裂和融合。

Scriptaid在猪体细胞核移植应用的研究进展

体细胞核移植(somatic cell nuclear transfer,SCNT)效率低下严重制约了克隆猪的产出,而在SCNT胚胎中表现出的表观遗传重编程(epigenetic reprogramming)不完全是导致该结果的重要原因。大量的研究表明组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor,HDACi),如Scriptaid(SCR),能够修复克隆胚胎的表观遗传重编程状态,包括提高供体核组蛋白的乙酰化水平、促进重要转录因子的转录活性以及增强染色质重塑等,从而促进SCNT胚胎的发育。本综述在参阅国内外研究成果的基础上对SCR在猪克隆中的应用进行分析和论述,以期为进一步在克隆猪的生产实践中加以应用奠定基础。

胚胎干细胞与雌性生殖细胞互相转化的研究进展

胚胎干细胞是全能性的干细胞,可以向3个胚层的细胞转化;雌性生殖细胞也被认为是一种"干细胞",因为其将遗传信息从一代传至下一代。两者具有相同和不同的特殊标志物,并且可以通过不同的方法互相转化,为"胚胎干细胞-卵母细胞环路"提出设想。

BoLA-Ⅰ类基因在牛体细胞克隆胚早期发育中的表达

体外培养成熟的卵母细胞,通过核移植(Nuclear transfer,NT)技术,构建体细胞克隆(Somatic cell nuclear transfer,SCNT)胚胎,以体外受精胚胎作为对照组。收集MⅡ期卵母细胞、2细胞胚胎、4细胞胚胎、8细胞胚胎、16细胞胚胎、桑椹胚和囊胚,采用实时荧光定量PCR的方法检测经典BoLA-I基因和非经典BoLA-I基因在MⅡ期卵母细胞及各期胚胎中的相对表达量。结果显示,BoLA-I类基因在在牛MⅡ期卵母细胞中的相对表达量显著高于其在其他各时期胚胎的相对表达量(P<0.05);BoLA-I类基因在牛SCNT胚和IVF胚中相对表达量随胚胎发育的变化趋势类似,随着第1次及随后的卵裂,经典BoLA-I、NC1、NC2基因mRNA的表达下降到极微的水平;NC3、NC4在整个发育阶段均表达很低甚至难以检测到它们表达。结果表明,本试验通过在体外制备及培养SCNT胚胎,初步建立BoLA-I类基因在其的表达模式,为BoLA-I类基因参与母胎免疫机制的研究奠定基础,为进一步寻找评估胚胎质量的标志物提供依据。