神经元限制性沉默因子在NTera2/CloneD1细胞向神经元分化模型中表达的检测

目的:建立NTera2/CloneD1细胞向神经元分化的模型,检测神经元限制性沉默因子(NRSF)经分化培养基诱导后表达的变化。方法:收集正常培养的NTera2/CloneD1细胞及经全反式维甲酸(RA)、阿糖胞苷(AraC)、尿苷分阶段诱导共28 d的细胞,显微镜下观察诱导前后细胞的形态学变化;免疫荧光法检测NTera2/CloneD1细胞诱导前后干性标志Nestin、Sox2和成熟神经元特异性标志NF-200、β-tubulinⅢ的表达情况;应用RT-PCR和免疫荧光法对NRSF进行mRNA和蛋白水平的检测。结果:显微镜下观察到正常培养的NTera2/CloneD1细胞呈克隆样生长,经分化培养基诱导后的NTera2/CloneD1细胞表现出典型的神经元样细胞形态。免疫荧光检测表明,未诱导的NTera2/CloneD1细胞表达神经干细胞的标志Sox2、Nestin,不表达成熟神经元特异性蛋白NF-200、β-tubulinⅢ;而经RA等诱导分化的细胞则不表达Sox2、Nestin,表达NF-200、β-tubulinⅢ。RT-PCR和免疫荧光检测显示,NRSF在诱导分化后的NTera2/CloneD1细胞中的表达量显著降低。结论:建立了NTera2/CloneD1细胞向神经元分化的模型,NRSF在诱导后的NTera2/CloneD1细胞中表达量显著下调,提示NTera2/CloneD1细胞在诱导过程中可能通过下调NRSF,使受到NRSF负性调控的神经元特异性蛋白启动表达并上调,进而实现NTera2/CloneD1细胞向神经元的定向分化。

RNA-Seq transcriptome analysis of porcine cloned and in vitro fertilized blastocysts

Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture.Whereas the approach remains inefficient and underlying mechanisms remain ambiguous.Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized（IVF） embryos before implantation,they appeared to have much lower full-term developmental efficiency in pig and cattle,and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them.Herein,RNA sequencing technique was used to screen differentially expressed genes in cloned and IVF blastocysts,and in total 628 differentially expressed transcripts were obtained,among which,280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts.Moreover,one statistically significant pathway associated with endoplasmic reticulum（ER） protein processing was enriched,and some ER-stress markers such as ATF4,ATF6,PDIA3,HSPA1 B,HSP40 and HSP90 between cloned and IVF blastocysts were suggested.Additionally,some developmentally important genes such as lipid metabolism related genes（MGLL,DDHD2 and FADS2） and epigenetic modification genes（DNMT1,KDM5 C and MBD3L5） were found differentially expressed between cloned and IVF embryos.

An Evaluation of the Infection Status and Source of Subgroup J Avian Leukosis Virus in Cloned Free-Range Layers

In recent years, subgroup J avian leukosis virus （ALV-J） has been found to frequently infect layers in China. This virus is responsible for economic losses due to both mortality and decreased performance in chickens. In this study, 45-d-old cloned flee-range layers were suspected to be infected with ALV and other immunosuppressive diseases because their feathers were unkempt and their growth rate was impaired. To estimate the infection status and determine the source of ALV-J in the flock, 30 cloacal swabs were randomly collected to measure the p27 antigen level by enzyme-linked immunosorbent assay （ELISA）. Among the birds that were tested, 87% （26/30） were positive. In addition, 6 anticoagulant blood samples were aseptically collected at random from the flock when the layers were 60 d old. These samples were centrifuged to obtain the leukocytes, which were then used to inoculate chicken embryo fibroblast （CEF） cells for the identification of ALV-J by indirect immunofluorescence （IFA）. Of the samples tested, 100% （6/6） were positive. The flock＇s production performance was also investigated, and 10 layers were necropsied to evaluate pathological changes at 115 d of age. The flock never laid eggs even though they reached the age of the first laying （110 d）. Furthermore, there were pathological changes present, including atrophy of the thymus and bursa of Fabricius, undeveloped ovaries, glandular stomach haemorrhage, and hepatosplenomegaly. Paraffin-embedded sections of intumescent liver and spleen were prepared for antigen localisation using IFA. Positive signals were prevalent in paraffin-embedded sections of the intumescent liver and spleen. Furthermore, provirus DNA was extracted from 4 cloned flee-range layers, and 2 patemal parents （HR native cocks）, and the gp85 gene of ALV-J was amplified by PCR to analyse the genetic variation. The results of the autogenous variation analysis showed that the 6 strains were 98.5-99.7% homologous. This study indicated that there was persistent infection with ALV-J by dynamic inspection, which seriously reduced the production performance of the flock. In addition, the genetic variation analysis showed that ALV-J in the flock was more likely to have originated from the paternal parent, the HR native cock.

Superovulation of the Cloned Cattle Derived from Somatic Cells and the Transfer of the Vitrified-Thawed Embryos of the Cloning Cattle

In this experiment, it was designed to carry out superovulation on the two cloned cattles, vitrification and transfer of the embryos recovered from them. First of all, it was carried out vitrification on embryos obtained by IVF. Results showed that there were no significant differences between the blastocysts (obtained by IVF) vitrified in EPS10 and these in EPS20 on the resuscitative rate and the developmental rate. The hatched rate of the blastocysts vitrified in EPS10 (31.3%, 35/112) was significantly higher than that in EPS20 (12.2%, 13/107)(P<0.01), so EPS20 was selected as the vitrification solution to freeze the embryos recovered from the cloned cattle. After superovulation, six (four usable embryos) and ten (nine usable embryos) embryos were respectively recovered from Kangkang and Shuanghuang. Two embryos were selected from the recovered embryos of each cloned cattle to freeze in EPS20, subsequently thawed and transferred into luteal ipsilateral uterine horns of 4 Holstein recipient cows after synchronization of estrus, respectively. At last, one recipient cow (No. 9908) became pregnant and delivered one healthy calf (descendant of the cloned cattle-Shuangshuang). The results of this experi- ment show that the cloned cattle as well as common cattle had better response to the exotic FSH and better ability to multiovulation, the embryos recovered from the cloned cattle can be vitrificated.

THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

Pathogenic Effects of Cloned Genomic DNA of Porcine Circovirus-like Virus P1 on Neonatal Mice via Different Inoculation Routes

[Objective] The paper was to explore the pathogenicity of cloned genomic DNA of porcine circovirus-like virus Pl to neonatal mice via different inoculation routes （brain, liver and muscle）. [Method] Cloned genomic DNA of P1 was inoculated to neonatal mice via different routes of brain, liver and muscle. Tissues of heart, liver, spleen, lung, kidney and brain were taken from neonatal mice at 7, 14 and 21 d post inoculation, re- spectively. Pl in various tissues were qualitatively and quantitatively detected by using ordinary PCR and quantitative real-time PCR. Meanwhile, histopathological changes were analyzed. [Result] Pl was detected in neonatal mice inoculated through three different routes. The viral load of tis- sues at 7 d post inoculation was significantly higher than those at 14 and 21 d post inoculation. Moreover, muscle inoculation led to the highest viral load in all tissues of neonatal mice. [Conclusion] Pl infection caused different degrees of pathological damage to heart, liver, lung, kidney and brain in neonatal mice.

Infectivity Analysis of Cloned Genomic DNA of P1 Agent in vitro

The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone had numbers of intracytoplasmic inclusions,and a few cells had intranuclear inclusions.Intracytoplasmic inclusions were round to oval and 0.1-0.3μm in diameter,and intranuclear inclusions,which were more electron dense,were of two general types:the first were round and small(0.1μm approximately)and the second were hexagonal and larger(0.4-0.8μm in diameter).Cells transfected with the tandem dimer of the P1 molecular DNA clone tested positive for P1 DNA at passage 5.This is the first report that the P1 molecular clone has infectivity in vitro and it will provide fundamental materials for further study of the biological characterization of P1.

Characteristics of nucleotides and amino acids in cDNA sequence of xyloglucan endotransglycosylase cloned from Anthocephalus chinensis

Xyloglucan endotransglycosylase is an identified relaxation factor with functions of easing and extending of plant cell walls.Its activities are directly related to plant growth and elongation of organisms.Anthocephalus chinensis is a tall and fast growing evergreen tree species.We cloned the full cDNA sequence of AcEXT genes which is abundantly expressed in the cambium of A.chinensis.The sequence analysis of nucleotides and amino acids revealed the presence of a 1396 bp full cDNA sequence,including a 960 bp complete open reading frame（ORF） encoding a 320 amino acid protein.The deduced amino acid sequence of AcXET was homologous to the other known XET proteins and contained the conserved EIDFE catalytic site which was specific to all the XETs.Our data should serve as a foundation for further insight into AcXET gene molecular mechanisms during wood formation and cell wall engineering of woody plants.

1B Specific LMW-GS Primers Cloned a 1D Located Gene from Wheat Cultivar Xiaoyan 22

In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.

Cloned goats(Capra hircus)from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μmol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells pre-cooled at 4℃ for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.

A framework for cloned vehicle detection

Rampant cloned vehicle offenses have caused great damage to transportation management as well as public safety and even the world economy.It necessitates an efficient detection mechanism to identify the vehicles with fake license plates accurately,and further explore the motives through discerning the behaviors of cloned vehicles.The ubiquitous inspection spots that deployed in the city have been collecting moving information of passing vehicles,which opens up a new opportunity for cloned vehicle detection.Existing detection methods cannot detect the cloned vehicle effectively due to that they use the fixed speed threshold.In this paper,we propose a two-phase framework,called CVDF,to detect cloned vehicles and discriminate behavior patterns of vehicles that use the same plate number.In the detection phase,cloned vehicles are identified based on speed thresholds extracted from historical trajectory and behavior abnormality analysis within the local neighborhood.In the behavior analysis phase,consider the traces of vehicles that uses the same license plate will be mixed together,we aim to differentiate the trajectories through matching degree-based clustering and then extract frequent temporal behavior patterns.The experimental results on the real-world data show that CVDF framework has high detection precision and could reveal cloned vehicles’behavior effectively.Our proposal provides a scientific basis for traffic management authority to solve the crime of cloned vehicle.

STUDIES ON A CLONED cDNA COMPLEMENTARY TO mRNA INDUCED BY TESTOSTERONE IN MOUSE KIDNEY CELLS

In the course of studying the hormonic regulation of gene expression, the DNA sequences complementary to the mRNA induced by testosterone from the mouse kidney cells was synthesized and made recombinant plasmids with pBR322. The cloned recombinant plasmid was used as a probe through molecular hybridization to detect the expression of the androgen inducible genes.

Comparison of birth weight and umbilical and placental characteristics of cloned and arti?cial insemination-derived piglets

Somatic cell nuclear transfer(SCNT)-derived piglets have signi?cantly higher stillbirth rate and postnatal mortality rate than arti?cial insemination(AI)-generated piglets. The question whether the low survival rate of SCNT piglets was related to birth weight, umbilical cord or placenta development was investigated. In this study,stillbirth rate, neonatal death rate, birth weight, umbilical cord status, placental parameters and placental gene expression patterns were compared between SCNT and AI piglets. Results showed that mortality rates at birth and during the neonatal stage of SCNT piglets were signi?-cantly higher than those of AI piglets. The incidence of abnormal umbilical cord in SCNT and SCNT-liveborn(SCNT-LB) piglets was signi?cantly higher than in AI and AI-liveborn(AI-LB) piglets. Birth weight, placental weight, placental surface area and placental ef?ciency in SCNT and SCNT-LB piglets were signi?cantly lower than those of AI and AI-LB piglets. Placental expression pro?les of imprinting, angiopoiesis and nutrient transportrelated genes were defective in SCNT-LB piglets compared with those in AI-LB piglets. Thus, the low survival rate of SCNT piglets may be associated with abnormal umbilical cord and placenta development. These characteristics may have resulted from aberrant expression of angiogenesis, nutrient transport, and imprinting-related genes in the placentas.

The ecological adaptability of cloned sheep to free-grazing in the Tengger Desert of Inner Mongolia,China

Since the birth of the first cloned sheep,somatic cell nuclear transfer technology has been successfully used to clone a variety of mammals.Cloned livestock have no apparent health risks,and the quality and safety of the cloned animal products are similar to non-cloned animals.The social behavior and environmental adaptability of postnatal cloned animals,especially when used for grassland farm production purposes,is unknown.In the present study,the cloned Dorper sheep equipped with GPS location devices were free-grazed in a harsh natural environment similar to conditions commonly experienced by Mongolian sheep.The main findings of this research were as follows.(1)Under free-grazing conditions,the cloned sheep showed excellent climatic and ecological adaptability.In extreme temperature conditions ranging from–30 to 40°C,the cloned sheep maintained acceptable body condition and behaved as other sheep.(2)The cloned sheep quickly adapted from a herd feeding strategy to the harsh environment and quickly exhibited a grazing regimen as other free-grazing sheep.(3)The cloned sheep exhibited free-grazing patterns and social behavior as other sheep.(4)The cloned sheep in the harsh environment thrived and produced healthy lambs.Overall,the cloned Dorper sheep exhibited excellent ecological adaptation,which is an important consideration for breeding meat sheep by cloning.The Dorper sheep readily adapted to the free-grazing conditions on the Mongolian plateau grassland,which attests to their ability to withstand harsh environmental conditions.

THE FIRST CLONED MONKEYS

Two cloned macaques named Zhong Zhong and Hua Hua play at the non-human-primate research facility under the Chinese Academy of Sciences in Suzhou,Jiangsu Province,on January 22.China announced it successful y cloned the world’s fi rst macaques from somatic cells on January 24.The development makes research with customizable populations of genetically uniform monkeys a possibility.

Cloned Sheep May Age Prematurely

1996年的头条科技新闻之一是:多利羊被克隆成功。世人曾为消息雀跃,以为克隆技术马上可以造福人类了,而且科幻作家也开始忙碌起来。而今,当多利羊过3岁生日时,人们却伤感地发现: In Dolly’s case,she is 3,but her genetic material is aging at the rate of the6-year-old sheep from which she was cloned. 这就是所谓aging prematurely。这则消息给人们带来的忧虑有两条。一是:被克隆的动物的预期寿命比人们想象的要短;二是:人们是否能够有效利用克隆的人体细胞去治疗疾病。目前,科学家们的担心还是集中于后者。本书收入的另一篇有关克隆的文章(It’s A Boy！Scientists Clone First Male Mammal)和本篇构成了强烈的对照,可谓一喜一忧。然而,无论喜忧,人类在克隆技术方面正在以坚实的步伐向前迈进。

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors; in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes; oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p>0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p<0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p>0.05). However, no statistical difference was observed between NTEs derived from oocytes of 36 h IVM group; NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p<0.05); blastocyst formation (9.3% vs. 13.2%, p<0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O2) tension; NTEs derived from oocytes matured under higher oxygen tension (20% O2) in cleavage rate (70.6% vs. 67.1%, p<0.05); blastocyst rate (11.8% vs. 12.3%, p<0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension; higher oxygen tension in IVM have no significant effect on development of cloned embryos.

Cloned goats (Gapra hircus) from foetal fibroblast cell lines

Mammalian cloning has been one of the most active research topics in the world. Cioning with in vitro culured foetal fibroblast cells, in comparison with embryonic cells, can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals, but also to utilize the unlimited fibroblast cells to produce large numbers of clonings. The preliminary results are as follows: (i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%, 77% and 35%, 31%, respectively. There is no significant statistical difference between them. (? These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast celi lines, which are the first cloned mammals from somatic cells in China. This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development, and a novel technique for the