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.

WPA1 encodes a vWA domain protein that regulates wheat plant architecture

Plant height,spike,leaf,stem and grain morphologies are key components of plant architecture and related to wheat yield.A wheat(Triticum aestivum L.)mutant,wpa1,displaying temperaturedependent pleiotropic developmental anomalies,was isolated.The WPA1 gene,encoding a von Willebrand factor type A(vWA)domain protein,was located on chromosome arm 7DS and isolated by map-based cloning.The functionality of WPA1 was validated by multiple independent EMS-induced mutants and gene editing.Phylogenetic analysis revealed that WPA1 is monocotyledon-specific in higher plants.The identification of WPA1 provides opportunity to study the temperature regulated wheat development and grain yield.

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.

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A:Impacts on rescue of sequence-modifying viruses

Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.

Genetic analysis and fine mapping of a grain size QTL in the smallgrain sterile rice line Zhuo201S

The development and application of the small-grain rice sterile line Zhuo201S(Z201S)has demonstrated its potential for mechanized hybrid rice seed production,leading to significant cost reductions.However,the molecular mechanism responsible for the small-grain size characteristic of Z201S remains unclear.In this study,we conducted a genetic analysis using near-isogenic lines constructed from Z210S,a small-grain rice sterile line,and R2115,a normal-grain variety.The results revealed that the small-grain trait in Z201S is governed by a single partially dominant gene which also enhances grain number.Through mapping,we localized the causal gene to the short arm of chromosome 2,within a 113 kb physical region delimited by the molecular markers S2-4-1 and LB63.Transgenic analysis and gene expression assays indicated LOC_Os02g14760 as the most likely candidate gene,suggesting that the small-grain size trait of Z201S is controlled by a novel locus that has not been previously identified.

Diagnostic and prognostic role of LINC01767 in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is a primary contributor to cancer-related mortality on a global scale.However,the underlying molecular mechanisms are still poorly understood.Long noncoding RNAs are emerging markers for HCC diagnosis,prognosis,and therapeutic target.No study of LINC01767 in HCC was published.AIM To conduct a multi-omics analysis to explore the roles of LINC01767 in HCC for the first time.METHODS DESeq2 Package was used to analyze different gene expressions.Receiver operating characteristic curves assessed the diagnostic performance.Kaplan-Meier univariate and Cox multivariate analyses were used to perform survival analysis.The least absolute shrinkage and selection operator(LASSO)-Cox was used to identify the prediction model.Subsequent to the validation of LINC01767 expression in HCC fresh frozen tissues through quantitative real time polymerase chain reaction,next generation sequencing was performed following LINC01767 over expression(GSE243371),and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes/Gene Set Enrichment Analysis/ingenuity pathway analysis was carried out.In vitro experiment in Huh7 cell was carried out.RESULTS LINC01767 was down-regulated in HCC with a log fold change=1.575 and was positively correlated with the cancer stemness.LINC01767 was a good diagnostic marker with area under the curve(AUC)[0.801,95% confidence interval(CI):0.751-0.852,P=0.0106]and an independent predictor for overall survival(OS)with hazard ratio=1.899(95%CI:1.01-3.58,P=0.048).LINC01767 nomogram model showed a satisfied performance.The top-ranked regulatory network analysis of LINC01767 showed the regulation of genes participating various pathways.LASSO regression identified the 9-genes model showing a more satisfied performance than 5-genes model to predict the OS with AUC>0.75.LINC01767 was down-expressed obviously in tumor than para-tumor tissues in our cohort as well as in cancer cell line;the over expression of LINC01767 inhibit cell proliferation and clone formation of Huh7 in vitro.CONCLUSION LINC01767 was an important tumor suppressor gene in HCC with good diagnostic and prognostic performance.

Gene Cloning and Bioinformatics Analysis of phoR Gene from Vibrio alginolyticus HY9901

PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain.A comprehensive analysis of the cloned gene was conducted using bioinformatics.Sequence analysis revealed that the total length of the phoR gene(GenBank accession No.:KJ958404.1)is 1299 bp,with the coding region containing a total of 432 amino acid residues.The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V.diabolicus.The SMART program was employed for the purpose of functional domain prediction,which revealed that PhoR possesses three major functional domains:PAS(amino acids 98-166),HisKA(amino acids 205-272),and HATPase_c(amino acids 317-429).

Cost-Effective Method of Gene Synthesis by Sequencing from Microchip-Derived Oligos for Droplet Cloning

Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.

Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.