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Sample records for bronchoepithelium egfp expression

  1. A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

    DEFF Research Database (Denmark)

    Klinck, Rasmus; Füchtbauer, Ernst-Martin; Ahnfelt-Rønne, Jonas;

    2011-01-01

    of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)(1Hri), to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates...

  2. Using the Tg(nrd:egfp/albino zebrafish line to characterize in vivo expression of neurod.

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    Jennifer L Thomas

    Full Text Available In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults.

  3. Using the Tg(nrd:egfp)/albino zebrafish line to characterize in vivo expression of neurod.

    Science.gov (United States)

    Thomas, Jennifer L; Ochocinska, Margaret J; Hitchcock, Peter F; Thummel, Ryan

    2012-01-01

    In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults.

  4. Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M M; Reynet, Christine; Schjerling, Peter

    2002-01-01

    the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase...... treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week......-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation...

  5. Establishment of a Tumor-bearing Mouse Model Stably Expressing EGFP Labeled Human MUC1 VNTRs

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shu-zi; ZHANG Hai-hong; ZHANG Wa; SHI He-liang; YU Xiang-hui; KONG Wei; LI Wei

    2008-01-01

    Two eukaryotic vectors expressing 9 tandem repeats of human MUCI(VNTR),VRI012-VNTR,and pEGFP-VNTR,were constructed by cloning VNTR gene into VR1012 and pEGFP,respectively,VNTR stably expressing murine Lewis lung carcinoma(LLC) cell Iine(VNTR+ LLC) was established by Lipofectamine-mediated transfection of pEGFP-VNTR into LLC cells,The EGFP expression was observed under a fluorescent microscope and VNTR expression in VNTR+ LLC cells was confirmed by means of Western blotting,A syngenic graft tumor model was generated by subcutaneous injection of VNTR+ LLC cells into C57/BL6 mice and tumor size increased rapidly with time and in a cell number dependent manner,VNTR mRNA expression in the tumor formed was confirmed by RT-PCR.After the third immunization mice were challenged subcutaneously with 5x10 5 VNTR* LLC cells,a significant reduction of subcutaneous tumor growth was observed in the groups immunized with VNTR plasmid DNA compared with that in the groups immunized with the vector DNA alone,Thus,the suppression of subcutaneous tumor was antigen-specific,This model is useful for the development of tumor vaccines targeting MUCI VNTRs.

  6. Stable EGFP Gene Expression in C6 Glioma Cell Line after Transduction with HIV-1-based Lentiviral Vector

    Institute of Scientific and Technical Information of China (English)

    JIN Gui-shan; LIU Fu-sheng; CHAI Qi; WANG Jian-jao; LI Jun-hua

    2008-01-01

    Objective:To establish a stable C6/EGFP glioma cell line for studies on glioma. Methods:The C6 glioma cell line was transfected with the human immunodeficiency virus type Ⅰ(HIV-1)based lentivirus vector containing two enhancer-promoters CMV and EF1α.Enhanced green fluorescent protein(EGFP)-positive C6 cells were sorted out by fluorescence-activated cell sort.Expression of EGFP was observed by fluorescent microscopy.EGFP gene in C6 genome was assessed by Polymerase chain reaction(PCR)and DNA sequencing.Original and transfected cells were compared biologically and cytomorphologically. Results:Lentivirus vector transfection produced up to 40% EGFP-positive cells.After fluorescence-activated cell sort selection,a pure cell line C6/EGFP was established.PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome.Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion:A C6/EGFP cell line expressing EGFP as a marker is established,in which the EGFP gene is integrated into the genome.This cell line can be served as a promising tool for further basic research and gene therapy studies.

  7. Effects of clinorotation on COL1A1- EGFP gene expression

    Institute of Scientific and Technical Information of China (English)

    DAI Zhongquan; LI Yinghui; DING Bai; ZHANG Yuguo; LIU Weiquan; LIU Pengpeng

    2004-01-01

    Bone-formation related gene plays a critical role in bone loss induced by space microgravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of α 1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vectors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the expression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.

  8. 小鼠pEGFP-Hoxa11真核表达载体的构建及表达%Construction of pEGFP-Hoxa11 Eukaryotic Expression Vector and Its Expression in CHO Cell Line

    Institute of Scientific and Technical Information of China (English)

    熊敏; 于倩; 崔爱娜; 张力; 朱桂金

    2011-01-01

    目的 构建和鉴定Hoxa11和EGFP双基因共表达真核载体.方法 采用DNA重组技术,将目的 基因Hoxa11克隆至含有报告基因EGFP的pEGFP-N1真核表达载体中,构建的真核表达载体pEGFP-Hoxa11经PCR,双酶切及基因测序鉴定;转染至CHO细胞,荧光显微镜下观察重组质粒的表达,提取细胞蛋白Western印迹检测蛋白表达.结果 pEGFP-Hoxa11重组质粒构建成功.构建的真核表达载体pEGFP-Hoxa11能在CHO细胞中有效表达.结论 成功构建了共表达Hoxa11和EGFP的真核表达载体,并能在CHO细胞中有效表达.为进一步研究Hoxa11的功能提供实验基础.%Objective To construct the recombinant plasmid pEGFP-Hoxa11 and detect its expression in CHO cell line. Methods The fragments of Hoxa11 was produced by PCR. After enzyme digestion by EcoRI and KpnI, the digested fragments were ligated into pEGFP vector overnight by T4 DNA ligase. The insertion of Hoxa11 in the recombinant plasmid of pEGFP-Hoxa11 was confirmed by PCR, enzyme digestion and DNA sequencing. The recombinant pEGFP-Hoxa11 was transfected into CHO cell lines. and EGFP-Hoxa11 expression was detected by fluorescence microscope and Western blotting analysis. Results The recombinant plasmid pEGFP-Hoxa11 was successfully constructed and its expression was visible in the transfected CHO cells under fluorescence microscope, and Hoxa11 expression was significantly increased in pEGFP-Hoxa11 transfection compared to the endogenous Hoxa11 level in empty vector transfected CHO cells. Conclusion The expression vector pEGFP-Hoxa11 was successfully constructed to co-express Hoxa11 and EGFP protein in CHO cell line.

  9. Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP.

    Science.gov (United States)

    da Silva, Joas L; Piuri, Mariana; Broussard, Gregory; Marinelli, Laura J; Bastos, Gisele M; Hirata, Rosario D C; Hatfull, Graham F; Hirata, Mario H

    2013-07-01

    Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.

  10. Optimization of PTS2-EGFP Expression in CHO and Vero Cells

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    Roozbeh Ghodratnama

    2007-01-01

    Full Text Available Objective: Reporter gene transfer to mammalian cells receives a great deal of attention due to its importance for molecular biology, embryology and developmental biology studies. Among DNA transfer technologies to eukaryotic cells, lipofection is known as the most widely used because of its easy handling procedure, low cell mortality and the natural pathway it undertakes.Materials and Methods: In this study we have examined the transfectability of two cell types: CHO and Vero cells via Lipofection in four different treatments, with combination of exposure duration, 3 and 6 hrs, and different plasmid DNA concentration, 0.5 and 1μgs. A fusion protein expression vector, pUcD2. PTS2-EGFP was used to direct the EGFP protein to peroxisomes after expression of related cDNA. An SPSS analysis was preformed after counting the positive cells.Results: optimum gene expression was found when using 1 μg DNA treated for three hrs for CHO cells, and 1 μg DNA treated for six hrs for Vero cells.Conclusion: The result suggests that CHO lipofection efficiency is significantly increased by both the DNA concentration and exposure time increment; however, an increase in exposure time has less significant effect on low DNA concentration conditions. The same results have been observed for Vero cells. Optimum expression was obtained with highest DNA concentration.

  11. The effect of housing temperature on the growth of CT26 tumor expressing fluorescent protein EGFP

    Science.gov (United States)

    Yuzhakova, Diana V.; Shirmanova, Marina V.; Lapkina, Irina V.; Serebrovskaya, Ekaterina O.; Lukyanov, Sergey A.; Zagaynova, Elena V.

    2016-04-01

    To date, the effect of housing temperature on tumor development in the immunocompetent mice has been studied on poorly immunogenic cancer models. Standard housing temperature 20-26°C was shown to cause chronic metabolic cold stress and promote tumor progression via suppression of the antitumor immune response, whereas a thermoneutral temperature 30-31°C was more preferable for normal metabolism of mice and inhibited tumor growth. Our work represents the first attempt to discover the potential effect of housing temperature on the development of highly immunogenic tumor. EGFP-expressing murine colon carcinoma CT26 generated in Balb/c mice was used as a tumor model. No statistically significant differences were shown in tumor incidences and growth rates at 20°C, 25°C and 30°C for non-modified CT26. Maintaining mice challenged with CT26-EGFP cells at 30°C led to complete inhibition of tumor development. In summary, we demonstrated that the housing temperature is important for the regulation of growth of highly immunogenic tumors in mice through antitumor immunity.

  12. Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2.

    Science.gov (United States)

    Berkova, Z; Morris, A P; Estes, M K

    2003-07-01

    The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.

  13. Rapid Construction of EGFP Labled Recombinant Adenovirus Containing hVEGF165 and Its Expression in Haematopoietic Cells

    Institute of Scientific and Technical Information of China (English)

    仲照东; 邹萍; 黄士昂; 胡中波; 刘凌波; 卢运萍

    2003-01-01

    By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labeled recombinant adenovirus vector containing hVEGF165 was constructed quickly and efficiently expressed in mouse haematopoietic cells. First, hVEGF165 coding sequence was subcloned into shuttle plasmid pAdTrack-CMV, then cotransformed with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF165. The expression of EGFP could be easily detected. The rate of EGFP positive mouse bone marrow mononuclear cells by flow cytometric analysis was 27.3 % (MOI= 100), and the expression of hVEGF165 protein in the supernatant was (1385+332) pg/106 cells. These results suggest that the construction of adenovirus vector by homologous recombination in bacteria features high efficiency and simplicity. The prepared high titer AdEGFP/hVEGF165 can be used an efficient helpful vector to infect hematopoietic cells.

  14. Effect of ploidy increase on transgene expression: example from Citrus diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene.

    Science.gov (United States)

    Xu, Shi-Xiao; Cai, Xiao-Dong; Tan, Bin; Li, Ding-Li; Guo, Wen-Wu

    2011-07-01

    Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy

  15. Potential utility of eGFP-expressing NOG mice (NOG-EGFP as a high purity cancer sampling system

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    Shima Kentaro

    2012-06-01

    Full Text Available Abstract Purpose It is still technically difficult to collect high purity cancer cells from tumor tissues, which contain noncancerous cells. We hypothesized that xenograft models of NOG mice expressing enhanced green fluorescent protein (eGFP, referred to as NOG-EGFP mice, may be useful for obtaining such high purity cancer cells for detailed molecular and cellular analyses. Methods Pancreato-biliary cancer cell lines were implanted subcutaneously to compare the tumorigenicity between NOG-EGFP mice and nonobese diabetic/severe combined immunodeficiency (NOD/SCID mice. To obtain high purity cancer cells, the subcutaneous tumors were harvested from the mice and enzymatically dissociated into single-cell suspensions. Then, the cells were sorted by fluorescence-activated cell sorting (FACS for separation of the host cells and the cancer cells. Thereafter, the contamination rate of host cells in collected cancer cells was quantified by using FACS analysis. The viability of cancer cells after FACS sorting was evaluated by cell culture and subsequent subcutaneous reimplantation in NOG-EGFP mice. Results The tumorigenicity of NOG-EGFP mice was significantly better than that of NOD/SCID mice in all of the analyzed cell lines (p  Conclusions This method provides a novel cancer sampling system for molecular and cellular analysis with high accuracy and should contribute to the development of personalized medicine.

  16. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

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    Veenhuis Marten

    2005-10-01

    Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of

  17. 大鼠pEGFP-C3/BMP-2真核表达载体的构建%Construction of rat pEGFP-C3/BMP-2 recombinant eukaryotic expressing vector

    Institute of Scientific and Technical Information of China (English)

    孙欣; 曾荣; 郭伟韬; 肖启贤; 王斌; 黄云; 林颢

    2012-01-01

    目的 通过克隆大鼠的BMP2基因,构建EGFP-C3/BMP2基因的真核细胞表达载体.方法 把大鼠的基因组DNA通过PCR获得BMP2,克隆构建载体pEGFP/C3-BMP2,并将其转化到大肠杆菌里面,最后进行重组真核表达载体pEGFP-C3-BMP2的构建和鉴定,并可观察其在真核细胞中的表达.结果 以大鼠总DNA为模板扩增出1 200 bp左右的特异性条带,测序结果与Gene-Bank测序结果相比,翻译成的氨基酸序列相同并完全一致,并可在真核细胞中表达.对重组质粒pEGFP-C3/BMP2进行双酶切鉴定并测序,结果也完全一致.结论 为进一步研究利用BMP2基因修饰骨组织工程骨,促进骨折愈合再生提供实验基础.%Objective To construct a recombinant eukaryotic expressing vector pEGFP-C3/BMP-2 by using rat bone morphogenetic protein 2 (BMP-2) gene clone. Methods BMP-2 was amplified with PCR and cloned into pEGFP-C3 vector after sequencing, recombinant eukaryotic expressing vector pEGFP-C3/BMP-2 was constructed and identified by sequencing, the expression of BMP-2 in eukaryotic cells was observed and analyzed. Results The sequencing of BMP-2 gene from the rat complied with the Gene-Bank result and with the same amino acid sequence after translation. The recombinant expressing vector pEGFP-C3/BMP-2 was confirmed by double enzyme digestion and sequencing, the successful expression of BMP-2 in eukaryotic cells was observed. Conclusion For the further study BMP2 genetic modification of bone tissue engineering, and promote the regeneration of fracture healing to provide the basis.

  18. 真核表达载体pIRES2-EGFP/TRAIL,pIRES2-EGFP/CD的构建和鉴定%Cloning and sequencing of reconstructed eukaryotic expression vector pIRES2-EGFP carrying CD and TRAIL genes

    Institute of Scientific and Technical Information of China (English)

    梁兵; 袁芳; 殷建瑞; 谭丽华; 高庆春; 高聪

    2011-01-01

    Objective To corstruct the eukaryotic expression vector encoding CD and TRAIL genes, pIRES2-EGFP/TRAIL and pIRES2-EGFP/CD, and provide the research basis of the association between overexpression of CD and TRAIL genes in C6 glioma cells.Methods The received plasmids DNA were assessed by electrophoresis in 1% agarose gel and sequencing.CD and TRAIL genes were cloned directionally into eukaryotic expression vector, pIRES2-EGFP, through double enzyme-cutting by Sac Ⅱ/Xho Ⅰ and BamH Ⅰ/Xho Ⅰ.The recombinant plasmids were converted into E.coli DH5α competent cell, and then bolted and identified by double enzyme-cutting of restriction enzyme, PCR, and nucleic acid sequence analysis.Results The length of two pIRES2-EGFP/TRAIL fragments after double-cutting by Sac Ⅱ/Xho Ⅰ were confident with theoretic length of 1.0 kb and 5.2 kb.The length of two pIRES2-EGFP/CD fragments after double-cutting by BamH Ⅰ/Xho Ⅰ were confident with theoretic length of 1.3 kb and 5.2 kb.pIRES2-EGFP/TRAIL and pIRES2-EGFP/CD were confirmed to be contained CD and TRAIL genes by PCR and nucleic acid sequence analysis.Conclusions Reconstructing pIRES2-EGFP/TRAIL and pIRES2-EGFP/CD could successfully establish abasis of further research of the association between overexpression of CD and TRAIL genes in C6 glioma cells.%目的 构建真核表达载体pIRES2-EGFP/TRAIL和pIRES2-EGFP/CD,为研究其联合表达对恶性胶质瘤的联合治疗作用提供基础.方法 将pCMV/CD质粒和pcDNA3.1(+)/TRAIL质粒行琼脂糖凝胶电泳检测,确定其完整性,并进行序列测定确定有无基因突变.pCMV/CD质粒用Sac Ⅱ/ Xho Ⅰ双酶切,pcDNA3.1(+)TRAIL质粒用BamH Ⅰ/XhoⅠ双酶切,将目的 基因定向克隆到真核细胞表达载体pIRES2-EGFP中,转化E.coli DH5α感受态细胞,通过限制性内切酶双酶切、PCR及核酸序列分析等筛选、鉴定重组质粒.结果 所构建的真核表达载体pIRES2-EGFP/TRAIL经Sac Ⅱ/ XhoⅠ双酶切回收片段分别为1

  19. Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Su Li; Chen Yunzhen; Zhang Xiaogang; She Qiang

    2005-01-01

    Objectives To construct a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expression in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFPC1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection.Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

  20. Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Di-Yong Xu; Xing Yao; Li-Shan Min; Ning Zhao; Bo-Ying Xu; Zheng-Ping Xu; Yong-Liang Lu

    2006-01-01

    AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells,respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

  1. Construction and identification of eukaryotic expression vector pIRES2-EGFP-hFasL%真核表达载体pIRES2-EGFP-hFasL的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    邱明链; 方航荣; 陈丽红; 刘景丰

    2011-01-01

    背景:FasL通过与靶细胞上Fas结合诱导程序性细胞死亡,可维持机体内稳态,诱导同种异基因移植免疫耐受,促进肿瘤细胞凋亡.目的:构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因hFasL的真核表达载体pIRES2-EGFP-hFasL.方法:通过实时聚合酶链反应RT-PCR方法从人外周血淋巴细胞中扩增出hFasL基因,与真核空载体 plRES2-EGFP一起,经XhoⅠ和EcoRⅠ双酶切,T4 DNA连接酶连接,从而构建pIRES2-EGFP-hFasL.用紫外分光光度计测定质粒浓度及纯度,经酶切、PCR技术、基因测序等方法进行鉴定.结果与结论:扩增出的hFasL条带大小约846 bp,构建的plRES2-EGFP-hFasL经酶切后在846 bp和2 000 bp处有特异性条带,DNA测序证实hFasL与Genebank上的序列完全一致.提示成功构建了含有hFasL及EGFP的真核表达载体plRES2-EGFP-hFasL.%BACKGROUND: FasL induced target cells to programmed cell death by binding with Fas, which was critically important to steady-state mechanism for the body, immune tolerance and tumor apoptosis mechanisms.OBJECTIVE: To construct the eukaryotic expression vector pIRES2-EGFP-h FasL containing enhanced green fluorescent protein report gene (EGFP) and target gene hFasL.METHODS: FasL gene was amplified from human peripheral blood lymphocytes by using real time polymerase chain reaction (RT-PCR), then pIRES2-EGFP-hFasL plasmid was constructed through the Xho I and EcoR I double digestion and T4 DNA ligase conjunction. The plasmid concentration and purity were detected by ultraviolet spectrophotometey and identified by endonuclease digestion, PCR and sequencing.RESULTS AND CONCLUSION: The amplified hFasL gene was about 846 bp in length. After digestion of recombinant plasmid pIRES2-EGFP-hFasL by restrictive enzymes, specific products with a size of 846 bp and 2000 bp were obtained. DNA sequencing indicates 100% coincidence between hFasL sequences of pIRES2-EGFP-hFasL and Genebank. These finding suggest that the

  2. Prokaryotic expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization antigen

    Institute of Scientific and Technical Information of China (English)

    Ping QU; Yanfang SUI; Libing LIU; Jiahai MA; Guangsheng CHEN; Jiankang CHEN; Fang'e LIU

    2008-01-01

    To study the endocytic activity of dendritic cells (DCs) by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood. Fusion protein HSP70-EGFP was prokaryotically expressed, isolated and puri-fied. DCs were isolated and cultured from human peri-pheral blood. The DCs were divided into 3 groups in the endocytic experiment. There were 106 DCs in each group. Group 1 and 2 were respectively incubated for 30 min. with HSP70-EGFP and EGFP. Group 3 was incubated with HSP70 for 30 min, and then incubated for 30 min. with HSP70-EGFP. Subsequently, 3 groups were placed in an incubator at 37℃ for 0.5, 1,2 and 24 h. Flow cytometry (FCM) was adopted to detect the amount of DCs with EGFP inside. IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12. There were 5 types in the experiment: LPS, inactive LPS, HSP70-EGFP, EGFP and no antigen. Fusion pro-tein HSP70-EGFP was successfully obtained and its molecular weight was 97 000. It accounted for 35.32% of the total protein. Under irradiation of an ultraviolet lamp, the protein solution sent out viridescent fluor-escence. The result detected by FCM indicated that after incubation for 0.5 h at 37℃, the positive rate in group 1 was 63%, while the other 2 groups were negative. After incubation for 1, 2 and 24 h at 37℃, the positive rates in the 3 groups were above 80%. The IL-12 Eli-spot exam-ination shows that with HSP70-EGFP being loaded, the amount of DCs secreting IL-12 was 134.09±31.78/105 cells, a little lower than that of DCs with LPS loaded (with the average point of 156.36±15.73). There was no significant difference between the 2 groups (P<0.01). By contrast, both of them were significantly higher than inactive LPS-(33.78±1.40)/105 cells and EGFP-loaded (23.13±4,57)/105 cells DC groups in the amount of DCs secreting IL-12 (P<0.01). The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs

  3. PiggyBac Transposon Mediated Efficient eGFP Expression in Porcine Somatic Cells and Cloned Embryos

    Institute of Scientific and Technical Information of China (English)

    Luo Yi-bo; Zhang Li; Zhu Jiang; Wu Mei-ling; Huan Yan-jun; Yin Zhi; Mu Yan-shuang; Xia Ping; LiuZhong-hua

    2012-01-01

    PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein (eGFP) and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer (SCNT), the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.

  4. 猪胰岛素启动子调控 EGFP 表达载体的优化及体外验证%Optimization and in vitro validation of EGFP expression controlled by porcine insulin promoter

    Institute of Scientific and Technical Information of China (English)

    于淑珍; 冯冲; 石宁宁; 宋小凤; 潘登科

    2014-01-01

    the first intron) to con-struct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP.Considering that the different location of restriction site may affect the expression efficien-cy of the transgene, we optimized the expression vector.Firstly the HindIII restriction site was deleted to realize the seam-less connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3′intron splicing acceptor site( SA) of the first intron into HindIII restriction site, named as PIP-SA( M)-EGFP.Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells.The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expres-sion efficiency of vectors.Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreaticβ-cells.RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with in-tron splicing.The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing.Mutation of the PIP splice site would cause the first in-tron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene.Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter.It provides the foundation for preparation of pigs with pancreaticβ-cells specifically expressing the transgene.

  5. Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells.

    Science.gov (United States)

    Chen, Hsin-Fu; Jan, Pey-Shynan; Kuo, Hung-Chih; Wu, Fang-Chun; Lan, Chen-Wei; Huang, Mei-Chi; Chien, Chung-Liang; Ho, Hong-Nerng

    2014-09-01

    Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4-BMP7-BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both.

  6. Ontogeny of CX3CR1-EGFP expressing cells unveil microglia as an integral component of the postnatal subventricular zone

    Directory of Open Access Journals (Sweden)

    Anna Lenice Ribeiro Xavier

    2015-02-01

    Full Text Available The full spectrum of cellular interactions within CNS neurogenic niches is still poorly understood. Only recently has the monocyte counterpart of the nervous system, the microglial cells, been described as an important cellular component in neurogenic niches. The present study sought to characterize the microglial population in the early postnatal subventricular zone (SVZ, the major site of postnatal neurogenesis, and in its anterior extension, the rostral migratory stream (RMS, a pathway for neuroblasts during their transit toward the olfactory bulb (OB layers. Analysis of the transgenic mice strain that has one of the locus of the constitutively expressed fractalkine CX3CR1 receptor replaced by the gene encoding the green fluorescent protein (EGFP circumvented the antigenic plasticity of the microglial cells. Here we show that common phenotypic markers of microglia do not reveal the full complement of these cells within the SVZ/RMS pathway. Remarkably, our analysis show that within the early SVZ/RMS pathway microglia are not proliferative and display a protracted development, retaining a more immature morphology than their counterparts outside germinal layers. Furthermore, during the first postnatal days microglia contact and phagocyte radial glia cells (RGs. Our results unveil microglial cells as a prominent component along the entire SVZ niche, together with neuroblasts and astroglial progenitors.

  7. Ontogeny of CX3CR1-EGFP expressing cells unveil microglia as an integral component of the postnatal subventricular zone.

    Science.gov (United States)

    Xavier, Anna L; Lima, Flavia R S; Nedergaard, Maiken; Menezes, João R L

    2015-01-01

    The full spectrum of cellular interactions within CNS neurogenic niches is still poorly understood. Only recently has the monocyte counterpart of the nervous system, the microglial cells, been described as an integral cellular component of neurogenic niches. The present study sought to characterize the microglia population in the early postnatal subventricular zone (SVZ), the major site of postnatal neurogenesis, as well as in its anterior extension, the rostral migratory stream (RMS), a pathway for neuroblasts during their transit toward the olfactory bulb (OB) layers. Here we show that microglia within the SVZ/RMS pathway are not revealed by phenotypic markers that characterize microglia in other regions. Analysis of the transgenic mice strain that has one locus of the constitutively expressed fractalkine CX3CR1 receptor replaced by the gene encoding the enhanced green fluorescent protein (EGFP) circumvented the antigenic plasticity of the microglia, thus allowing us to depict microglia within the SVZ/RMS pathway during early development. Notably, microglia within the early SVZ/RMS are not proliferative and display a protracted development, retaining a more immature morphology than their counterparts outside germinal layers. Furthermore, microglia contact and phagocyte radial glia cells (RG) processes, thereby playing a role on the astroglial transformation that putative stem cells within the SVZ niche undergo during the first postnatal days.

  8. Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

    Science.gov (United States)

    Mousai, M; Hosseini, S M; Hajian, M; Jafarpour, F; Asgari, V; Forouzanfar, M; Nasr-Esfahani, M H

    2015-10-01

    Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

  9. Dissecting the Ca²⁺ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells.

    Science.gov (United States)

    Díaz, Yuleima; Peña, Franshelle; Aristimuño, Olga Carolina; Matteo, Lorena; De Agrela, Marisela; Chemello, Maria Elena; Michelangeli, Fabian; Ruiz, Marie Christine

    2012-08-01

    Rotavirus infection modifies Ca(2+) homeostasis provoking an increase in Ca(2+) permeation, cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), total Ca(2+) pools and, a decrease of Ca(2+) response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca(2+) homeostasis is not yet known. It has been proposed that the increase in [Ca(2+)](cyto) is the result of Ca(2+) release from intracellular stores, thereby activating store-operated Ca(2+) entry (SOCE). We studied the mechanisms involved in the changes of Ca(2+) permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca(2+) stores induced by thapsigargin or ATP was not able to elicit Ca(2+) entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca(2+)](cyto), is permeable to Mn(2+) and inhibited by La(3+) and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca(2+) entry is small and not modified by infection. The plasma membrane permeability to Ca(2+) in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na(+)/Ca(2+) exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca(2+) entry.

  10. Construction and expression of recombinant plasmid pENTR-CMV-EGFP -hsa-mir-16-1/15a%重组质粒pENTR-CMV-EGFP-hsa-mir-16-1/15a的构建与表达

    Institute of Scientific and Technical Information of China (English)

    方潇碧; 张春鸿; 黄亚; 林森; 黄振校; 吴丽萍; 施清圆; 李文峰; 廖志苏

    2011-01-01

    目的:构建针对人鼻咽癌CNE-2Z细胞Bcl-2基因pENTR-CMV-EGFP-hsa-mir-16.1/15a真核表达质粒,转染至CNE-2Z细胞并检测其表达.方法:采用PCR法从重组质粒PGH-16-1/15a中获得16-1/15a-X2370G全长序列,在T4 DNA Ligase连接酶作用下连接入重组载体pENTR-CMV-EGFP.重组质粒经酶切及测序鉴定.将构建成功的重组质粒转染入人鼻咽癌细胞株CNE-2Z,用荧光显微镜观察转染结果.结果:重组质粒pENTR-CMV-EGFP-hsa-mir-16-1/15a经酶切与测序证实构建成功,转染至鼻咽癌CNE-2Z细胞后,荧光显微镜观察证实该重组质粒能在CNE-2Z中表达.结论:成功构建真核表达质粒DENTR-CMV-EGFP-hsa-mir-16-1/15a,并在鼻咽癌CNE-2Z细胞中得到表达,可用于进一步检测其抗肿瘤机制.%Objective To construct the eukaryotic expression plasmid pENTR-CMV-EGFP-hsa-mir-16-1/15a of human Bcl-2 gene,transfect it into nasopharyngeal carcinoma cells CNE-2Z and detect its expression.Methods The full-length cDNA of 16-1/15a-X2370G gene was amplified by PCR from the recombinant plasmid PCH-16-1/15a, then was linked with the recombinant vector pENTR-CMV-EGFP by T4 DNA Ligase. The recomhinant plasmid was identified by restriction enzyme digestion and sequence analvsis. Then the plasmid was correctly transfected into NPC cell CNE-2Z , and the effect of transfection was directly observed under fluorescence microscope. Results The green fluorescent protein was mainly detectd in the CNE-2Z cell , which suggested that the recomhinant plasmid was constructed successfully. Conclusion The eukaryotic expression plasmid pENTR-CMV-EGFP-hsa-mir-I6-1/15a was constructed successfully and expressed in NPC cell CNE-2Z. Its mechanism of antitumor will be further investigated.

  11. The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

    Science.gov (United States)

    Lewis, Jo E.; Brameld, John M.; Hill, Phil; Barrett, Perry; Ebling, Francis J.P.; Jethwa, Preeti H.

    2015-01-01

    Introduction The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. New method To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Results Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. Comparison with old method The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. Conclusion The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. PMID:26300182

  12. Construction and Expression of Prokaryotic Expression Vector pET28a-EGFP%原核表达载体pET28a-EGFP的构建与表达

    Institute of Scientific and Technical Information of China (English)

    季爱加; 宁喜斌

    2011-01-01

    An enhanced green fluorescent protein ( ECFP) gene fragment in plasmid PEGFP-N3 as a template was used to amplify and obtained the EGFP gene fragment by PCR, and then designed a primer to introduce EcoR I and Hind Ⅲ Bites lo its both ends. After treated with double restriction enzyme, the introduced enzymatic site ECFP gene fragment, and pET28a plasmid, a recombinant expression plasmid pET28a-EGFP was obtained uung Tt ligase. The pET28a-EGFP was transformed into competence cells of E. Coli BL21 with heal shock method. The inducer of isopro-pyl β-D-l-thiogalactopyranoside (IPTG) was added to induce EGFP expression, as the optical density under 600 nm OD600 =0.4 of the B. Coli LB (Luria-Bertani) culture medium. The results indicated that the recombinant plasmtd enzymatic sites characterization and sequencing was correct. Under the natural light, the transformant colonies assumed green in LB solid medium (contains 1 mmol/L IPTG and SO μg/mL kanamycin ( Kan) ). When excited with blue-ray under fluorescence microscope, these recombinants emitting green fluorescence could clearly be observed. The successfully constructed prokaryotic expression vector pET28a-EGFP that expressed effectively in E. Coli BL21 will provide certain theoretical and technical supports for marking food borne pathogens as fluorescent markers in the future.%以质粒PEGFP-N3中增强型绿色荧光蛋白(Enhanced Green Fluorescent protein,EGFP)基因片段为模板,利用PCR技术扩增得到EGFP基因片段,并设计引物在其2端引入酶切位点EcoRⅠ和HindⅢ,对引入酶切位点的EGFP片段和pET28a质拉进行双酶切处理后,利用T4连接醇连接得到了重组质粒pET28a-EGFP.利用热击法把得到的重组质粒pET28a-EGFP特化至E.coli BL21( Escherichia coli BL21)感受态细胞中,当大肠埃希菌LB(Luria-Bertani)培养液在600 nm下的光密度值OD600 =0.4时,通过添加异丙基硫代β-D-半乳糖苷(IPTG)作为诱导剂诱导EGFP表达.结果表明:重组质

  13. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    NARCIS (Netherlands)

    Blecha, Andreas; Zarschler, Kristof; Sjollema, Klaas A.; Veenhuis, Marten; Rödel, Gerhard; Rodel, G.

    2005-01-01

    Background: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested

  14. 不同EGFP-蜘蛛丝融合基因在昆虫细胞的表达及融合丝蛋白形成机制初探%Insights Into Formation Mechanism of the Silk Fusion Protein From Expression of Different Sizes of EGFP-spider Silk Fusion Protein in Insect Cells

    Institute of Scientific and Technical Information of China (English)

    赵爱春; 夏庆友; 鲁成; 向仲怀; 张袁松; 中垣雅雄

    2007-01-01

    蜘蛛能吐出具有优异机械特性的多种类型的丝,其中管状腺丝用于构建卵囊并具有良好的分子和机械性能,是目前唯一报道的全长cDNA 序列的蜘蛛丝.基于为将来利用生物工程方法大量生产高性能的仿蜘蛛丝纤维提供基础信息,通过Bac-to-Bac/AcNPV杆状病毒表达系统,在强启动子驱动下,对2个大小不同的蜘蛛卵囊丝蛋白基因序列(全长cDNA序列和部分cDNA序列)与EGFP报告基因实现了在AcNPV昆虫细胞中的融合表达.绿色荧光和Western印迹分析表明,被表达出的2个大小不同的卵囊丝融合蛋白在昆虫细胞胞质中呈现出明显不同的溶解性.含有部分卵囊丝基因的短EGFP-蜘蛛丝融合基因的表达产物在胞质里具有可溶性;而含有全长卵囊丝基因的巨大EGFP-蜘蛛丝融合基因的表达产物倾向于自我装配形成沉淀聚合物.研究结果也暗示了蜘蛛丝蛋白的分子大小可能对其装配成高性能的丝纤维有重要影响.%Spiders spin multiple types of silks that are renowned for their superb mechanical properties. Cylindrical silk with novel molecular and mechanical properties, used in the construction of egg case outercover, is only one type of spider silk full-length cDNA reported data to date. In order to provide some valuable messages for large-scale mimicking the outstanding high-performance properties of native spider silk by bioengineering method in the future, two different sizes of cylindrical silk protein genes (full-length and partial cDNA sequences) and EGFP fusion genes were expressed in insect cells under the control of strong promoter by Bac-to-Bac/AcNPV baculovirus expression system. EGFP fluorescence and western blotting analysis showed two different sizes of the expressed egg case silk fusion protein presented obviously different solubility within the aqueous cytosol of insect cells. The expression products of partial egg case silk fusion gene were soluble in aqueous

  15. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  16. Developmental dynamics of a polyhomeotic-EGFP fusion in vivo.

    Science.gov (United States)

    Netter, S; Faucheux, M; Théodore, L

    2001-08-01

    Polyhomeotic is a member of the Polycomb group of genes. The products of this group are chromatin-associated proteins that act together as multimeric complexes. These proteins are required for the maintenance of target gene repression in a permanent and heritable manner during development. In order to better understand the dynamics of their action during development, we generated transgenic flies expressing a polyhomeotic protein tagged with the enhanced green fluorescent protein. Here we show that this fusion protein (PH-EGFP) retains both the functional properties of the endogenous protein and its target specificity on polytene chromosomes. The distribution of the PH-EGFP protein is partly dependent on the presence of wildtype Polycomb protein, indicating that PH-EGFP behaves as does the wildtype PH protein. Therefore, the PH-EGFP chimera appears to be an appropriate reporter of PH protein distribution and a suitable tool for the study of Polycomb-group complex assembly in vivo. The subnuclear distribution of PH-EGFP is dynamic throughout development. In the interphase nucleus at the cellular blastoderm, a diffuse granular pattern is observed. From the early gastrula stage onward, a few brighter dots appear. As development progressed from germ band retraction through hatching of the larva, numerous discrete dots accumulate in the nucleus of epidermal cells. The increasing number of dots observed during development may indicate that PH-EGFP is recruited at different stages on different target sites, a result that is in good agreement with functional data previously reported.

  17. Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor

    Institute of Scientific and Technical Information of China (English)

    Hua JIANG; Jie ZHANG; Bi-zhi SHI; Yu-hong XU; Zong-hai LI; Jian-ren GU

    2007-01-01

    Aim: To develop a simple method for monitoring protein localization of epidermal growth factor (EGF) in living cells. Methods: Enhanced green fluorescent protein (EGFP) was used as an autofluorescent tag to label EGF ligands. SDS-PAGE and Western blot analysis were used to detect the expression of the EGFP-tagged EGF (EGFP-EGF) protein. The cell-binding and internalization activity of EGFP-EGF were analyzed by fluorescence-activated cell sorting (FACS) and confocal micro-scopy. Results: EGFP-EGF protein was expressed in Escherichia coil and purified.A cell-binding assay demonstrated that the EGFP-EGF protein could bind effi-ciently to the cells expressing EGFR. The binding and intemalization of EGFP-EGF can be visualized even at a very low concentration under confocal microscopy.The FACS-based assay for internalization activity indicated the accumulation of internalized EGFP-EGF over time. Furthermore, the results of the competition assay indicated its EGFR binding specificity. Using such a method, it does not need to label EGF with chemicals and avoid light in the experimental process. Conclusion: The fusion protein EGFP-EGF has several characters including high sensitivity, stability and convenience for manipulation, and is a powerful tool for the study of EGF endocytosis.

  18. Construction and Expression of Recombinant Adenovirus Vector Ad5-hTRX-EGFP%重组腺病毒载体Ad5-hTRX-EGFP的构建及其表达

    Institute of Scientific and Technical Information of China (English)

    扈江伟; 王军; 徐曼; 苏永锋; 孔维霞; 盛红霞; 张斌; 陈虎

    2012-01-01

    -defective recombinant adenovirus pAd-hTRX-EGFP was co-transfected in HEK293 cells, purified by CsCl gradient centrifugation, counted for virus particles and determined for tiler. The recombinant adenovirus was identified by PCR. The HEK293 cells were then transfected with adenoviruses and assayed by flow cytometry. The expression of hTRX was confirmed by Western blot. The results showed that according to PCR and restriction endonuclease assay, the target gene was inserted into recombinant adenovirus vector successfully. The sequence of fusion gene was the same as that of designed fragments. The titer of the purified recombinant adenovirus pAd-hTRX-EGFP was 5. 558 × 1010pfu/mL A transfection efficiency of 92. 25% could be achieved at MOI = 100. Western blot further confirmed that hTRX was efficiently expressed in HEK293 cells. It is concluded that recombinant adenovirus vector containing hTRX has been constructed successfully and obtained highly efficient virus that can express efficiently in HEK293 cells, which laid a foundation for further investigation.

  19. Study on construction of recombinant plasmid pIRESneo-EGFP-BDNF and its expression in bone mesenchymal stem cell in mice%重组质粒pIRESneo-EGFP-BDNF构建及转染骨髓间充质干细胞

    Institute of Scientific and Technical Information of China (English)

    张平; 赵钢勇; 陈凯; 宋月平; 康增军; 苏立凯

    2012-01-01

    目的 探讨重组质粒pIRESneo-EGFP-BDNF构建及转染至骨髓间充质干细胞(MSCs)制备BDNF基因工程细胞的方法.方法 将pEGFP(N1)-BDNF质粒进行改造与pIRESneo相连结,构建携带BDNF的高拷贝质粒pIRESneo-EGFP-BDNF,采用电转染技术转染骨髓MSCs,经G418筛选,通过倒置荧光显微镜判断转染效率,采用Western blot方式判定转染细胞是否表达BDNF蛋白.结果 经过双酶切鉴定,pIRESneo-EGFP-BDNF携带EGFP及BDNF基因,以EGFP为报告基因,质粒构建成功;通过电穿孔技术以及G418筛选,提高pIRESneo-EGFP-BDNF转染骨髓MSCs效率.结论 成功制备高效表达携带BDNF基因的质粒,且转染骨髓MSCs,为进一步开展BDNF基因治疗神经系统变性疾病奠定基础.

  20. 外源报告基因EGFP在盐藻中实现瞬时表达%Transient Expression of Reporter Gene EGFP in Transformed Dunaliella salina

    Institute of Scientific and Technical Information of China (English)

    李杰; 任宏伟; 黄洁虹; 俞梅敏; 茹炳根

    2003-01-01

    探索杜氏盐藻(Dunaliella salina)的转基因方法和筛选方法.利用烟草花叶病毒启动子(CaMV 35S)、衣藻叶绿体atpA启动子与来源于水母的加强型绿色荧光蛋白报告基因(EGFP)构建表达载体pART7GFP和pUCGFP,转化盐藻.EGFP在CaMV 35S启动下表达出绿色荧光蛋白,在荧光显微镜下看到发绿色荧光的转基因盐藻.根据荧光数目进行统计,转化效率高于5%.衣藻来源的启动子atpA在盐藻中未能启动EGFP的表达.用直径1 μm的金粉颗粒和0.6 μm的金粉进行基因枪法转化,1 μm的金粉颗粒成功将外源基因导入盐藻,用0.6 μm的金粉配合多种技术参数也没有将外源基因导入盐藻.EGFP可以用作盐藻遗传转化的报告基因使单细胞真核生物盐藻可以利用流式细胞术(FACS)等技术进行筛选,从而避开平板筛选转基因盐藻的限制,并使转基因盐藻实现无抗生素筛选成为可能.

  1. pVLT-EGFP载体构建及其在巴西固氮螺菌(Azospirillum brasilense)的表达研究%Construction of an Expression Vector pVLT-EGFP and Its Expression in Azospirillum brasilense

    Institute of Scientific and Technical Information of China (English)

    巩涛; 王继雯; 杨文玲; 甄静; 刘莹莹; 李冠杰; 刘莉; 岳丹丹; 陈国参

    2015-01-01

    为了研究巴西固氮螺菌(Azospirillum brasilense)在植物体内的定殖,利用酶切连接的方法,以增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因与表达栽体pVLT-33为基本元件,构建了重组表达载体pVLT-EGFP,电转巴西固氮螺菌R7细胞,并利用实时荧光定量PCR(qPCR)研究了不同温度、不同时间EGFP mRNA的表达情况.酶切及测序结果表明,成功构建了pVLT-EGFP载体,并在荧光显微镜下观察到绿色荧光蛋白表达;qPCR结果显示:30℃,诱导9hEGFP基因的表达水平最高.本研究成功构建了重组表达载体pVLT-EGFP,为实现pVLT-EGFP的可控表达及研究固氮菌在植物体内的定殖规律及促生长机理提供了一种有效的途径.

  2. Construction of ECM1 in eukaryotic expression vector and its expression in hepatocellular carcinoma cell line SMMC-7721%pEGFP-N2-ECM1重组质粒的构建及其在人肝癌细胞系SMMC-7721中的表达

    Institute of Scientific and Technical Information of China (English)

    董训忠; 李建生; 许戈良; 荚卫东; 陈浩; 任维华

    2012-01-01

    To establish the hepatocellular carcinoma( HCC ) cell line SMMC-7721 with stabilized expression of extracellular matrix protein l( ECM1 ). Methods The complete ECM1 gene amplificated from ECM1 cDNA by PCR was connected into pEGFP-N2 vector by PCR and DNA gene recombinant technique. The recombi-nant plasmid was detected by restrictive enzyme digestion and gene sequencing analysis; Sequenced right plasmid was transfected into SMMC-7721 cells using lipofectamine(tm)2000 and ECM1 highly expressed cells were selected with G418. ECM1 gene expression was detected by RT-PCR and the expression of fusion proteins ( GFP and ECM1 ) were observed under the fluorescence microscope. Results The construction of expression vector was accomplished. The SMMC-7721 cell line expressed stably ECM1 which was screened out. Conclusion The eukaryotic expression vector pEGFP-N2-ECMl is constructed and expressed stably in SMMC-7721 cells, which forms an important basis for the further studies of ECM1 in the HCC progression .%目的 构建细胞外基质蛋白-1(ECM1)稳定表达的SMMC-7721肝癌细胞系.方法 应用PCR和DNA重组技术构建pEGFP-N2-ECM1真核表达载体,经酶切、测序鉴定正确后,用脂质体转染SMMC-7721细胞,G418药物筛选稳定转染的细胞系.荧光显微镜检测融合蛋白表达,RT-PCR技术检测ECM1基因表达.结果 构建的重组质粒经双酶切及测序分析鉴定,结果 证实pEGFP-N2-ECM1构建成功;筛选获得稳定表达ECM1的SMMC-7721细胞.结论 成功构建了pEGFP-N2-ECM1的真核表达载体,并在肝癌SMMC-7721细胞中稳定表达,为研究ECM1在肝癌进展中的作用奠定了实验基础.

  3. Expression of Recombinant pEGFP-N3-APC Vectors in HT-29 Colorectal Carcinoma Cell Line%重组质粒pEGFP-N3-APC在结肠癌细胞株HT-29中的表达

    Institute of Scientific and Technical Information of China (English)

    吕梁; 霍继荣; 刘斌; 刘佳; 武捷; 王捷

    2008-01-01

    [目的]构建含有APC蛋白功能区域的pEGFP-N3-APC重组质粒,转染结肠癌细胞HT-29,观察重组质粒的表达.[方法]设计引物分别扩增5条APC基因功能区域片段.将扩增片段与pEGFP-N3载体连接后挑取阳性克隆,行菌落PCR和测序鉴定.使用Lipofectamine 2000将重组质粒转染结肠癌细胞株HT-29,观察细胞中绿色荧光蛋白的表达.[结果]构建5条带有APC不同结构域重组真核表达载体pEGFP-N3-APC,重组真核表达载体转染HT-29细胞后,可观察到绿色荧光蛋白的表达.[结论]真核细胞表达载体pEGFP-N3-APC的成功构建,为进一步研究其在细胞内的功能提供了基础.

  4. A Csf1r-EGFP Transgene Provides a Novel Marker for Monocyte Subsets in Sheep.

    Science.gov (United States)

    Pridans, Clare; Davis, Gemma M; Sauter, Kristin A; Lisowski, Zofia M; Corripio-Miyar, Yolanda; Raper, Anna; Lefevre, Lucas; Young, Rachel; McCulloch, Mary E; Lillico, Simon; Milne, Elspeth; Whitelaw, Bruce; Hume, David A

    2016-09-15

    Expression of Csf1r in adults is restricted to cells of the macrophage lineage. Transgenic reporters based upon the Csf1r locus require inclusion of the highly conserved Fms-intronic regulatory element for expression. We have created Csf1r-EGFP transgenic sheep via lentiviral transgenesis of a construct containing elements of the mouse Fms-intronic regulatory element and Csf1r promoter. Committed bone marrow macrophage precursors and blood monocytes express EGFP in these animals. Sheep monocytes were divided into three populations, similar to classical, intermediate, and nonclassical monocytes in humans, based upon CD14 and CD16 expression. All expressed EGFP, with increased levels in the nonclassical subset. Because Csf1r expression coincides with the earliest commitment to the macrophage lineage, Csf1r-EGFP bone marrow provides a tool for studying the earliest events in myelopoiesis using the sheep as a model.

  5. Construction of pEGFP-N3-APC vectors carrying various APC functional domains and their expression in HCT-II6 cells%APC蛋白功能区域重组质粒的构建及其在结肠癌细胞株HCT-116中的表达

    Institute of Scientific and Technical Information of China (English)

    吕梁; 霍继荣; 刘佳; 张宏斌; 武捷; 王捷

    2009-01-01

    目的 构建并鉴定含有APC蛋白不同功能区域的真核细胞表达载体pEGFP-N3-APC1~5,转染人类结直肠癌细胞HCT-116,观察重组质粒在细胞内的表达.方法 根据APC基因的功能结构以及APC突变簇集区的特点,设计特异性引物扩增APC基因特异性的功能区域片段.将扩增出的5个APC片段克隆到pEGFP-N3载体的N端,经测序分析对重组质粒pEGFP-N3-APC1~5进行鉴定.使用脂质体转染法将重组质粒转染人类结直肠癌细胞HCT-116,通过观察细胞中绿色荧光蛋白的表达情况来检测APC功能区域在细胞内的表达.以RT-PCR法进一步验证重组质粒在细胞中的表达.结果 构建5个pEGFP-N3-APC结构区域的重组真核表达载体,重组真核表达载体转染HCT-116细胞后,细胞中均可见绿色荧光蛋白的表达.RT-PCR结果显示,5个蕈组质粒在HCT-116细胞中均可表达.结论 真核细胞表达载体pEGFP-N3-APC1~5的成功构建,为进一步研究其在细胞内的功能,筛选结直肠癌基因治疗的有效且易于基因操作的靶标片段提供了基础.%Objective To construct recombinant plasmids containing various functional domains of APC protein and detect their expression in HCT-116 cells. Methods Five APC gene fragments were amplified by PCR with whole APC gene as template and primers designed according to APC cDNA sequence and mutation cluster domain. The five obtained fragments were cloned into eukaryotic expression vector pEGFP-N3 to generate recombinant pEGFP-N3-APC1-5. Sequence of the inserted gene was identified and analyzed after restriction enzyme digestion. Liposome-mediated recombinant plasmid pEGFP-N3-APC was transfected into HCT 116 cells and identified by green fluorescence. RT-PCR was employed to validate the expression of recombinant vectors in cells. Results Recombinant pEGFP-N3-APC1-5 were confirmed by restriction enzyme digestion and sequence analysis. The plasmids could be expressed in HCT-116 cell line

  6. pHSP70P-EGFP真核表达载体的构建及其在人Chang's肝细胞中的表达%Construction of eukaryotic expression vector pHSP70P-EGFP and its expression in human Chang's liver cell

    Institute of Scientific and Technical Information of China (English)

    高峰; 陈亚军; 杨学文

    2011-01-01

    目的 构建HSP70启动子与绿色荧光蛋白重组真核表达载体,并在人Chang's肝细胞表达,建立一种新的体外药物肝毒性早期预测细胞模型.方法 提取人Chang's肝细胞基因组DNA,钓取HSP70启动子基因,构建重组载体,经脂质体转染人Chang's肝细胞,用G418筛选稳定转染细胞,用荧光显微镜观察绿色荧光表达情况,用实时荧光定量PCR检测EGFP基因表达量的变化.结果 成功钓取HSP70启动子基因并导入真核表达载体pEGFP-N1;G418以300 ng/mL的终浓度筛选出稳定转染的单克隆细胞;荧光显微镜观察到肝毒性药物酮康唑刺激人Chang's 肝细胞后,可诱导HSP70启动子调控增强型绿色荧光蛋白发出绿色荧光,药物刺激组EGFP mRNA相对表达量与正常对照组比较有统计学意义(t=-14.21,P<0.05).结论 成功构建HSP70启动子与绿色荧光蛋白共表达真核表达载体,获得稳定转染单克隆细胞,并证实HSP70在肝毒性药物刺激下的应激表达,为建立新的体外药物肝毒性早期预测细胞模型进行高通量筛选新药奠定了基础.%Objective To construct the eukaryotic expression vector of HSP70 promoter and green fluorescent protein and expression the recombine proteins in human Chang's liver cells in order to establish a new drugs hepatotoxicity cell model in vitro for early prediction. Method The genome DNA of human Chang's liver cell was extracted and the promoter of HSP70 gene was obtained. HSP70 promoter gene was transfected to human Chang's liver cell by means of pEGFP-Nl vector with liposome. The steadily transfected cells were screened by G418 selection. The expression of green fluorescent protein was observed by fluorescence microscope. The change of the expression of EGFP gene were observed by real - time fluorescence quantitative PCR. Results pEGFP-Nl vector expressing recombi-nant HSP70 promoter and green fluorescent protein was successfully constructed and the steadily transfected

  7. Characterization of functionally active interleukin-18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells.

    Science.gov (United States)

    Wu, Hsing Chieh; Chen, Yu San; Shien, Jui Hung; Shen, Pin Chun; Lee, Long Huw

    2016-05-01

    The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL-18a cell line that stably expresses the fusion protein chIL-18 was constructed and the enhanced green fluorescence protein connected through a (G4 S)3 linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL-18a cells (1 × 10(5) ) were inoculated in 60-mm culture dishes containing 5 mL of media to achieve 50%-60% confluence and were cultured in the presence of the cycle-specific inhibitors 10058-F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058-F4, aphidicolin, and colchicine, respectively, the aphidicolin-induced single cells showed higher fusion protein levels than did the 10058-F4- or colchicine-induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin-treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN-γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581-591, 2016.

  8. The dark side of EGFP: defective polyubiquitination.

    Directory of Open Access Journals (Sweden)

    Mathijs Baens

    Full Text Available Enhanced Green Fluorescent Protein (EGFP is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-kappaB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered.

  9. The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis.

    Science.gov (United States)

    Li, Wei; Ding, He; Zhang, Xinxin; Cao, Lili; Li, Jianhua; Gong, Pengtao; Li, He; Zhang, Guocai; Li, Shuhong; Zhang, Xichen

    2012-03-01

    Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis.

  10. 核基质结合区修饰的附着体载体介导的EGFP基因体外表达%MAR-Modified Episomal Vector-Mediated EGFP Expression in Vitro

    Institute of Scientific and Technical Information of China (English)

    梅文瀚; 吴兆平; 徐荣婷; 钱关祥; 卢健

    2006-01-01

    目的 建立一个非病毒介导外源基因高效转移和稳定表达体系.方法 采用EB病毒(EBV)来源的附着体质粒载体,并结合顺式作用元件IFN-MAR构建重组质粒pcDNA3-EGFP-EBVR和pcDNA3-EGFP-EBNA1-MAR.采用荧光显微镜观察并摄片,FACS及逆转录-聚合酶链反应检测EGFP表达阳性的COS-7细胞比例、荧光表达强度及EGFP基因的转录.通过Southern印迹和质粒还原实验分析质粒DNA在细胞中存在的形式.结果 本实验构建的重组质粒载体pcDNA3-EGFP-EBVR和pcDNA3-EGFP-EBNA1-MAR在转染进入COS-7细胞后以染色体外附着体的形式存在.由于EBVR的存在,可以在体外获得EGFP较长时间的稳定表达.IFN-MAR替代OriP后,仍可在体外获得EGFP的稳定表达.结论 MAR修饰的EBV附着体载体可以在体外获得外源基因高效稳定的表达.

  11. Construction and Expression of the Polycistronic Vector of Avian Polyomavirus 1 Late Genes Tagged with EGFP%禽类多瘤病毒APV-1晚期基因多顺反子的EGFP标记载体构建和表达

    Institute of Scientific and Technical Information of China (English)

    李劲; 刘琳

    2016-01-01

    为研究禽类多瘤病毒晚期基因多顺反子翻译起始调控的机制,设计和构建了以增强绿色荧光蛋白( EGFP)报告基因替代病毒APV-1野生型的晚期结构蛋白VPs基因的真核双顺反子表达载体,并观察其在转染进入禽类原代成纤维细胞中的表达翻译状态。以APV-1的cDNA克隆(pHL1003)为模板,运用PCR技术扩增出与野生型病毒APV-1相同的Ori区、早期编码区和晚期Agno-1a区序列作为载体片段;从质粒pEGFP-N1中扩增出编码绿色荧光的EGFP DNA片段;经连接构成重组质粒pHL1003-GFP,通过脂质体细胞转染方法将质粒DNA转染到鸡胚胎和鹌鹑胚胎成纤维细胞中,通过细胞培养观察荧光表达状况。 DNA序列分析证实了重组克隆中的EGFP序列与已知的质粒pEGFP-N1中EGFP序列一致。转染72 h后观察鸡和鹌鹑胚胎成纤维细胞,转染成功胚胎细胞明显表达较强的荧光,说明GFP可以在构建的双顺反子重组质粒的下游中正常表达并产生荧光。 pHL1003-GFP重组质粒的构建及其在禽类原代成纤维细胞中的高效表达,为我们研究多顺反子翻译起始调控中Agno-1a基因的表达对下游病毒结构蛋白基因的翻译调控机制提供了简洁易用的系统和模型。%To study the mechanism of translational initiation regulation of the polycistronic mRNA of the Avain Polyomavirus late genes, the eukaryotic expression vector with bi-cistronic mRNA was designed and constructed, in which the downstream late structural proteins of wild-type APV-1 were replaced by EGFP reporter gene. By transfection of the recombinant DNA into the bird primary fibroblast cells, the expression of EGFP was observed. Using APV-1 cDNA plasmid as template, the early and Ori regions including Agno-1a gene of the viral genome were amplified by PCR. The EGFP coding sequence was amplified from plasmid pEGFP-N1. After ligation of the above two kinds of DNA fragments, the recombinant plasmid pHL1003-GFP

  12. In vitro cultivation of rat bone marrow mesenchymal stem cells and establishment of pEGFP/Ang-1 transfection method

    Directory of Open Access Journals (Sweden)

    Xiu-Qun Zhang

    2014-09-01

    Conclusions: Adherence screening method and density gradient centrifugation can be effective methods to obtain BMSCs with high purity and rapid proliferation. Besides, the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.

  13. Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaoxia [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); University of Chinese Academy of Sciences, Beijing (China); Chen, Xiaowen; Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Ningbo Laboratory, State Key Laboratory of Freshwater Ecology and Biotechnology (China)

    2014-07-01

    The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.

  14. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masahito [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Umeyama, Kazuhiro [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); International Cluster for Bio-Resource Research, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Matsunari, Hitomi [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Takayanagi, Shuko [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Nakauchi, Hiromitsu [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  15. Construction of eukaryotic fusion expression vectors containing CIDE-3 with EGFP and DsRed 1 and determination of their expressions and localization in 293T cells%EGFP-CIDE-3及DsRed 1-CIDE-3融合基因真核表达载体的构建及其在293T细胞中的表达和定位

    Institute of Scientific and Technical Information of China (English)

    谷雨; 李青; 叶菁; 李烦繁; 闵婕; 张丽英; 马钰; 李航; 刘芳

    2008-01-01

    目的:分别构建EGFP、DsRed1与CIDE-3的融合基因真核表达载体,观察其在人胚肾上皮细胞293T中的表达,确定CIDE-3的亚细胞定位. 方法:分别以质粒pEGFP-C3、pDsRed 1-N1及本实验室克隆得到的质粒pET28a(+)-CIDE-3为模板,PCR扩增EGFP、DsRed 1 DNA片段及人CIDE-3基因的CDS序列,再分别将EGFP、CIDE-3及DsRed 1、CIDE-3 克隆人真核表达载体pShuttle-CMV中.酶切、测序鉴定后,经磷酸钙转染人293T细胞,通过荧光显微镜观察其在293T细胞中的表达,并利用荧光染料Bodipy 493/503定位脂滴,探讨CIDE-3与脂滴之间的关系.结果:酶切及DNA测序证实,重组质粒pShuttle-CMV-EGFP-CIDE-3和pShuttle-CMV-DsRed 1-CIDE-3构建成功.荧光显微镜观察显示,pShuttle-CMV-EGFP-CIDE-3融合蛋白定位于细胞质,pShuttle-CMV-DsRed 1-CIDE-3融合蛋白也定位于细胞质,并与脂滴存在共定位关系. 结论:成功构建了重组质粒pShuttle-CMV-EGFP-CIDE-3和pShutde-CMV-DsRed 1-CIDE-3;两者均可在239T细胞中表达,融合蛋白分布于细胞质,并与脂滴存在共定位关系.

  16. 牛TLR2全长基因表达质粒的构建及其在HEK293细胞中的表达(英文)%Construction of an Expression Plasmid pEGFP-N1-boTLR2 for Full-length Bovine TLR2 and Its Expression in HEK293 Cells

    Institute of Scientific and Technical Information of China (English)

    王玉明; 王静萱

    2012-01-01

    [Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.%[目的]构建牛TLR2全长基因表达质粒,并在HEK293细胞中表达。[方法]利用RT-PCR技术克隆TLR2基因的全长编码区,连接到pMD18-Tsimplevector,再亚克隆到pEGFP-N1载体,得到包含TLR2基因全长的重组真核表达质粒。将重组质粒瞬时转染到HEK293细胞。流式细胞计数法和共聚焦显微镜法检测转染效率和表达蛋白在细胞中的定位;qRT-PCR法检测TLR2 mRNA在HEK293/boTLR2中的表达。最后,通过脂膜酸刺激HEK293/boTLR2细胞试验来分析TLR2蛋白的生物活性。[结果]成功克隆TLR2基因全长并连接到真核表达载体,并在HEK293细胞中表达。在LTA刺激的条件下,转染重

  17. Transformation of Beauveria bassiana to produce EGFP in Tenebrio molitor for use as animal feed additives.

    Science.gov (United States)

    Kim, Jae Su; Choi, Jae Young; Lee, Se Jin; Lee, Ju Hyun; Fu, Zhenli; Skinner, Margaret; Parker, Bruce L; Je, Yeon Ho

    2013-07-01

    Efforts are underway to develop more effective and safer animal feed additives. Entomopathogenic fungi can be considered practical expression platforms of functional genes within insects which have been used as animal feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by highly infective Beauveria bassiana ERL1170. Among seven test isolates, ERL1170 treatment showed 57.1% and 98.3% mortality of mealworms 2 and 5 days after infection, respectively. The fungal transformation vector, pABeG containing the egfp gene, was inserted into the genomic DNA of ERL1170 using the restriction enzyme-mediated integration method. This resulted in the generation of the transformant, Bb-egfp#3, which showed the highest level of fluorescence. Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-days mealworm sections showed a strong fluorescence. This did not occur in the wild-type strain. This work suggests that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.

  18. Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

    Directory of Open Access Journals (Sweden)

    Longquan Quan

    Full Text Available Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs. The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT. The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.

  19. Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology.

    Science.gov (United States)

    Reichenbach, Myriam; Lim, Tiongti; Reichenbach, Horst-Dieter; Guengoer, Tuna; Habermann, Felix A; Matthiesen, Marieke; Hofmann, Andreas; Weber, Frank; Zerbe, Holm; Grupp, Thomas; Sinowatz, Fred; Pfeifer, Alexander; Wolf, Eckhard

    2010-08-01

    Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.

  20. Trichomonas vaginalis virus-mediated expression of EGFP in the parasitic protozoan Trichomonas vagi-nails%阴道毛滴虫病毒介导的EGFP在阴道毛滴虫细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    郭瑞娟; 李淑红; 张西臣; 李建华; 宫鹏涛; 杨举; 张国才

    2010-01-01

    目的 研究阴道毛滴虫病毒(Trichomonas vaginalis virus,TVV)介导外源基因进入阴道毛滴虫体内表达的能力,探索TVV作为双链RNA病毒转染载体的可能性.方法 根据TVV基因组的序列特征,用绿色荧光蛋白(EG-FP)编码基因替换TVV的全部或部分基因编码区,构建TVV与增强型EGFP编码基因的嵌合体pTVV-EGFP,其体外转录体经电穿孔方法转染携病毒阴道毛滴虫株,RT-PCR及SDS-PAGE方法检测EGFP的表达情况.结果 电穿孔转染后培养的虫体在荧光显微镜下观察到绿色荧光信号,且续传15代后仍然存在;RT-PCR检测到EGFP的mRNA,SDS-PAGE检测到转染后虫体及培养上清中有分子质量单位为27 ku的蛋白,与已知EGFP的分子质量相符.结论 TVV能成功介导外源性EGFP编码基因在阴道毛滴虫体内表达.

  1. Nr4a1-eGFP is a marker of striosome-matrix architecture, development and activity in the extended striatum.

    Directory of Open Access Journals (Sweden)

    Margaret I Davis

    Full Text Available Transgenic mice expressing eGFP under population specific promoters are widely used in neuroscience to identify specific subsets of neurons in situ and as sensors of neuronal activity in vivo. Mice expressing eGFP from a bacterial artificial chromosome under the Nr4a1 promoter have high expression within the basal ganglia, particularly within the striosome compartments and striatal-like regions of the extended amygdala (bed nucleus of the stria terminalis, striatal fundus, central amygdaloid nucleus and intercalated cells. Grossly, eGFP expression is inverse to the matrix marker calbindin 28K and overlaps with mu-opioid receptor immunoreactivity in the striatum. This pattern of expression is similar to Drd1, but not Drd2, dopamine receptor driven eGFP expression in structures targeted by medium spiny neuron afferents. Striosomal expression is strong developmentally where Nr4a1-eGFP expression overlaps with Drd1, TrkB, tyrosine hydroxylase and phospho-ERK, but not phospho-CREB, immunoreactivity in "dopamine islands". Exposure of adolescent mice to methylphenidate resulted in an increase in eGFP in both compartments in the dorsolateral striatum but eGFP expression remained brighter in the striosomes. To address the role of activity in Nr4a1-eGFP expression, primary striatal cultures were prepared from neonatal mice and treated with forskolin, BDNF, SKF-83822 or high extracellular potassium and eGFP was measured fluorometrically in lysates. eGFP was induced in both neurons and contaminating glia in response to forskolin but SKF-83822, brain derived neurotrophic factor and depolarization increased eGFP in neuronal-like cells selectively. High levels of eGFP were primarily associated with Drd1+ neurons in vitro detected by immunofluorescence; however ∼15% of the brightly expressing cells contained punctate met-enkephalin immunoreactivity. The Nr4a1-GFP mouse strain will be a useful model for examining the connectivity, physiology, activity and

  2. Construction of Prokaryotic Expression Plasmid pET15b-YARA-EGFP and Expression and Purification of YARAEGFP Fusion Protein%pET15b-YARA-EGFP原核表达质粒的构建及融合蛋白YARA-EGFP的表达与纯化

    Institute of Scientific and Technical Information of China (English)

    陈思思; 王家宁; 黄永章; 郭凌郧; 郑飞

    2009-01-01

    目的:构建原核表达载体 pET15b-YARA-EGFP,并进行YARA-EGFP融合蛋白的表达和纯化.方法:用分子克隆技术构建出表达型载体 pET15b-YARA-EGFP,在E.coli BL21(DE3)中表达融合蛋白YARA-EGFP,并进行Ni2+-NTA树脂柱亲和层析以纯化蛋白.结果:经测序证实成功构建了表达型载体pET15b-YARA-EGFP,YARA-EGFP融合蛋白在E.coli BL21(DE3)中得到表达,纯化后的蛋白浓度为1.098 mg/mL.SDS-PAGE和Western blot分析表明纯化蛋白为目的蛋白YARA-EGFP.结论:已成功制备出YARA-EGFP融合蛋白.

  3. Expression of Truncated uPA Fused to EGFP in HEK293F Cells%人尿激酶型纤溶酶原激活因子截短型突变体与绿色荧光蛋白在真核细胞HEK293F中的融合表达

    Institute of Scientific and Technical Information of China (English)

    张连成; 高丽华; 张昕; 潘芸; 高招刚; 李伊培; 胡显文; 陈惠鹏

    2013-01-01

      Objective: To construct and express eukaryotic expression vectors of the truncated urokinase-type plas⁃minogen activator(uPA) fused to enhanced green fluorescent protein(EGFP). Methods: EGFP and truncated uPA genes were amplified by PCR using plasmid pIRES2-EGFP and recombinant plasmid pcDNA3.1(+)/uPA as tem⁃plates, and inserted into eukaryotic expression vector pcDNA3.1(+) sequentially. The constructed recombinant plas⁃mids were transfected into HEK293F cells, and treated with high concentration G418. The expression of recombi⁃nant proteins was detected by confocal microscopy and ELISA. Results: DNA sequencing proved that the eukaryot⁃ic expression vectors of the fusion proteins were constructed successfully. And the green fluorescent protein could be observed in cells by confocal microscopy after the transfection, and the stable expression cell lines were got af⁃ter selected by G418. ELISA showed that the secreting type fusion proteins exist in supernatant. Conclusion: Re⁃combinant plasmids have been constructed and expressed in HEK293T cells, which will contribute to further re⁃search of the interaction of uPA and its biological function in cells.%  目的:构建人尿激酶型纤溶酶原激活因子(uPA)截短型突变体与绿色荧光蛋白(EGFP)分泌型融合表达载体并在真核细胞中表达。方法:采用PCR法,分别以质粒pIRES2-EGFP和重组质粒pcDNA3.1(+)/uPA为模板,扩增出带BamHⅠ和XbaⅠ酶切位点的EGFP及带NheⅠ和HindⅢ酶切位点的uPA截短体基因片段,先后将EGFP和截短型uPA基因片段克隆到真核表达载体pcDNA3.1(+)上,转入HEK293F细胞,用G418对转染细胞进行加压筛选,通过共聚焦显微镜观察和ELISA方法鉴定表达产物。结果:DNA测序结果显示,uPA不同截短型突变体基因片段与EGFP基因融合的真核表达载体构建成功,共聚焦显微镜观察发现HEK293F细胞中有绿色荧光且定位

  4. Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Cheng [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Graduate University of Chinese Academy of Sciences, Beijing (China); Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)

    2012-07-15

    Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.

  5. An E.coil SOS-EGFP biosensor for fast and sensitive detection of DNA damaging agents

    Institute of Scientific and Technical Information of China (English)

    Zhilan Chen; Meiling Lu; Dandan Zou; Hailin Wang

    2012-01-01

    An E.coli SOS-EGFP biosensor which expresses enhanced green fluorescent protein as a reporter protein under the control of recA gene promoter in SOS response was constructed for detection of DNA damage and evaluation of DNA damaging chemicals.The chemicals that may cause substantial DNA damage will trigger SOS response in the constructed bacterial biosensor,and then the reporter egfp gene under the control of recA promoter is stimulated to express as a fluorescent protein,allowing fast and sensitive fluorescence detection.Interestingly,this biosensor can be simultaneously applied for evaluation of genotoxicity and cytotoxicity.The SOS-EGFP bacterial biosensor provides a sensitive,specific and simple method for detecting known and potential DNA damaging chemicals.

  6. Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.

    Directory of Open Access Journals (Sweden)

    Luis Vidali

    Full Text Available BACKGROUND: Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. METHODOLOGY/PRINCIPAL FINDINGS: In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. CONCLUSIONS/SIGNIFICANCE: Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.

  7. Construction of EGFP-tagged rBCG of E.tenella and distribution in chickens

    Institute of Scientific and Technical Information of China (English)

    WANG QiuYue; LI JianHua; ZHANG XiChen; LIU ChengWu; CAO LiLi; REN KeYan; GONG PengTao; CAI YaNan

    2009-01-01

    Chicken coccidiosis is a major parasitic disease with substantial economic burden to the poultry in-dustry. Enhanced green fluorescent protein (EGFP) tagged recombinant Bacille Calmette-Guerin (rBCG), as a fusion protein with coccidian rhomboid antigen was constructed to track rBCG in vivo in chickens in this study. Immunization of chickens with one dose of rBCG pMV361-Rho/EGFP induced humoral immune response. The colonization of rBCG in liver, spleen, lung, kidney and caecum was observed by laser confocal microscopy. Real-time quantitative RT-PCR showed s rise expression level of rhomboid protein on the 7th day and a peak on the 14th day and disappearance on the 28th day after immunization. These results have significant implications for the development of rBCG vaccines against avian coccidiosis.

  8. Development of Neutralization Assay Using an eGFP Chikungunya Virus

    Directory of Open Access Journals (Sweden)

    Cheng-Lin Deng

    2016-06-01

    Full Text Available Chikungunya virus (CHIKV, a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV with an isolated strain (assigned to Asian lineage from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.

  9. Construction of EGFP-tagged rBCG of E.tenella and distribution in chickens

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Chicken coccidiosis is a major parasitic disease with substantial economic burden to the poultry industry.Enhanced green fluorescent protein(EGFP) tagged recombinant Bacille Calmette-Guerin(rBCG),as a fusion protein with coccidian rhomboid antigen was constructed to track rBCG in vivo in chickens in this study.Immunization of chickens with one dose of rBCG pMV361-Rho/EGFP induced humoral immune response.The colonization of rBCG in liver,spleen,lung,kidney and caecum was observed by laser confocal microscopy.Real-time quantitative RT-PCR showed a rise expression level of rhomboid protein on the 7th day and a peak on the 14th day and disappearance on the 28th day after immunization.These results have significant implications for the development of rBCG vaccines against avian coccidiosis.

  10. Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation.

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    Taku Saito

    Full Text Available Induced pluripotent stem cells (iPSC are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

  11. 外源EGFP表达对肿瘤细胞体外生物学行为的影响%Exogenous EGFP labeling affects tumor cells biological behavior

    Institute of Scientific and Technical Information of China (English)

    张敏; 赵文晶; 冯海凉; 刘艳艳; 李占稳; 刘玉琴

    2012-01-01

    目的 探讨常用的可视化细胞标记技术-外源EGFP表达对肿瘤细胞生物学行为的影响.方法 选择不同的携带EGFP的载体,利用脂质体转染或慢病毒感染技术,在不同肿瘤细胞系中表达外源EGFP,筛选稳定表达外源EGFP的细胞系,MTT法检测细胞体外增殖能力;细胞分析计数仪(CasyTT型)检测细胞大小;Transwell体外迁移实验检测细胞体外侵袭能力.结果 成功建立了稳定表达外源EGFP的人结肠癌细胞系HCT116-GFP、HCT116-EGFP、COLO320DM-EGFP和小鼠树突状细胞肉瘤细胞系DG6-EGFP.HCT116-EGFP细胞、COLO320DM-EGFP细胞和DG6-EGFP细胞的体外增殖能力明显降低;HCT116-EGFP细胞体积增大:平均直径分别为(14.53±0.07) μm( HCT116)和(18.28±0.16) μm (HCT116-EGFP) (P<0.01);HCT116-EGFP细胞的体外侵袭能力明显降低(P<0.01).结论 外源EGFP可视化标记细胞后,可能会影响肿瘤细胞的生物特性,利用这些模型进行科研时,需注意对相关指标的影响.%Objective To study the influence of exogenous EGFP labeling on biological behavior of tumor cells in vitro. Methods Using liposome transfection or lentivirus infection, exogenous EGFP carried by different vectors was expressed in different cell lines. The exogenous EGFP expressing cell lines were selected for further analysis. The growth was measured by MTT assay. The distribution of cell size was tested by cell counter( CasyTT). The in-" vasion was measured by Boyden-chamber assay. Results Human colon cancer cell lines HCT116-GFP, HCT116-1 EGFP, COLO320DM-EGFP and murine dendritic cell sarcoma cell line DG6-EGFP with stable expression of exoge-nous EGFP were successfully established. After expression of exogenous EGFP, the growth of HCT116-EGFP cells, COLO320DM-EGFP cells and DG6-EGFP cells significantly decreased. The cell volume of HCT116-EGFP cells was bigger than that of HCT116 cells. The invasive ability of HCT116-EGFP cells was significantly decreased

  12. Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.

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    Johan Seijsing

    Full Text Available Studies on the neonatal Fc receptor (FcRn have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

  13. Construction and expression of recombinant plasmid TAZ-pcDNA3 .1 and TAZ-pEGFP-C2%T AZ 基因重组质粒的构建与表达

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    仲念念; 朱伶俐; 王旋; 房娜

    2015-01-01

    Objective Two recombinant plasmids , TAZ‐pcDNA3 .1 and TAZ‐pEGFP‐C2 , were established . The protein expression of TAZ in HEK293 cells was detected by Western Blot and the roles of TAZ in promoting cell proliferation and migration were further explored . Methods AZ gene was amplified by PCR , fragments were recovered followed by connection with glue T carrier , blue‐white screening , transformation and extraction of plasmid DNA . Then the plasmid DNA was digested , connected by T 4 DNA Ligase , and then sub‐cloned into pEGFP‐C2 and pcDNA3 .1 to construct new recombinant plasmids . These plasmids were transfected into HEK293 cells to observe the distribution of TAZ using a fluorescence detector . The protein expression was detected by Western Blot .Results By restriction enzyme identification and sequence analysis , the recombinant plasmids were successfully constructed . Fluorescent photos show that the distribution of TAZ molecule was in the nucleus and cytoplasm . Western Blot test results showed that TAZ molecule could induce over‐expression of specific proteins . Conclusion Two recombinant plasmids were successfully constructed . The effects of TAZ over‐expression were validated , which will lay a foundation for revealing the mechanism of TAZ in promoting cell proliferation and migration .%目的:构建重组质粒TAZ‐pcDNA31.及 TAZ‐pEGFP‐C2,并应用Western Blot检测TAZ蛋白在细胞内的表达情况,初步探索TAZ分子促进细胞增殖和迁移的作用机制。方法通过PCR扩增获得 TAZ基因片段,胶回收后连接T载体,蓝白斑筛选,转化,提质粒,酶切,用T4 DNA Ligase连接,亚克隆进入pEGFP‐C2和pcDNA31.获得新的重组质粒,分别转染 HEK293细胞,智能型荧光细胞监测仪观察TAZ分子在细胞内的分布情况,Western Blot检测其在细胞内的表达情况。结果重组质粒经双酶切鉴定和测序证明构建成功,荧光照片显示 TAZ分子分布在

  14. The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

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    Wang Y

    2014-10-01

    Full Text Available Yun Wang,1 Fu-xing Lin,2 Yu Zhao,1 Mo-zhen Wang,2 Xue-wu Ge,2 Zheng-xing Gong,1 Dan-dan Bao,1 Yu-fang Gu1 1Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University, 2CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China Abstract: Novel submicron core-shell-structured chitosan-based composite particles ­encapsulated with enhanced green fluorescent protein plasmids (pEGFP were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC. pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. Keywords: gene therapy, gene transfection, hydroxybutyl chitosan, thiolated N-alkylated chitosan, pEGFP, complex coacervation

  15. Adenoviral-mediated Hath1-EGFP gene transfer into guinea pig cochlea through intact round window membrane

    Institute of Scientific and Technical Information of China (English)

    CHEN Wei; HU Yin-yan; YANG Shi-ming; GUO Wei; SUN Jian-he; HAN Dong-yi; ZHAI Suo-qiang; YANG Wei-yan; David Z.Z.He

    2008-01-01

    Objective To study expression of adenovira1-mediated Hathl-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane (RWM), and to assess its effects on hearing. Methods Twenty adult guinea pigs were used, of which: 12 were surgically inoculated with AdHath1-EGFP in the bony groove of round window niche, and 8 with artificial perilymph. Auditory brainstem response(ABR) thresholds were determined in all animals before and 5 days after surgery. On post-surgery day 5 and day 14, animals were sacrificed and whole mounts of cochlea and fro zensections were examined. Results ABR tests showed no significant change of hearing after the surgery.Strong fluorescence staining in the cochleae was seen in Ad-Hathl-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with tittle decrease on post-surgery day 14.The contralateral cochlea and those in the control groups were free of fluorescence staining. Conclusion The transgenic Hath1-EGFP can be effectively delivered into the inner ear through intact RWM, in an atraumatic manner.

  16. Construction of Eukaryotic Expression Vector pCB1-EGFP and its Expression in HeLa Cells%大鼠大麻素Ⅰ型受体绿色荧光融合蛋白真核表达载体的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    冯睿; 范娟; 刘一辉; 钱召强; 魏春玲; 任维

    2014-01-01

    目的:构建大鼠大麻素型Ⅰ受体绿色荧光融合蛋白真核表达载体并观察其在细胞中的表达.方法:大鼠CB1基因序列设计引物,以大鼠脑组织为模板扩增CB1基因编码区片段,克隆至增强型绿色荧光蛋白表达载体pEGFP-N3中,构建重组融合蛋白表达载体pCB1-EGFP.将pCB1-EGFP质粒转染HeLa细胞,通过观察EGFP报告基因的表达以及免疫荧光,Western Blot方法鉴定CB1可在真核细胞中过表达情况.结果:构建重组融合蛋白表达载体pCB 1-EGFP,单双酶切和测序验证正确.将pCB1-EGFP质粒转染HeLa细胞,荧光显微镜下观察到融合表达的绿色荧光蛋白,且呈胞膜表达.免疫荧光试验也证明重组载体转染后,CB1基因和GFP共同定位于胞膜部分.Western Blot实验证明表达CB1蛋白.结论:成功构建了高表达的CB 1-EGFP融合蛋白真核表达载体.

  17. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  18. In vitro cultivation of rat bone marrow mesenchymal stem cells and establishment of pEGFP/Ang-1 transfection method

    Institute of Scientific and Technical Information of China (English)

    Xiu-Qun Zhang; Long Wang; Shu-Li Zhao; Wei Xu

    2014-01-01

    Objective:To obtain the bone marrow mesenchymal stem cells (BMSCs), complete phenotypic identification and successfully transfect rat BMSCs by recombinant plasmid pEGFP/Ang-1. Methods:BMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method, and purified. Then the recombinant plasmid pEGFP/Ang-1 was used to transfect BMSCs and the positive clones were obtained by the screen of G418 and observed under light microscopy inversely. Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1. Results: BMSCs cell lines were obtained successfully by adherence screening method and density gradient centrifugation. Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs, which can express Ang-1 for 3 d and decreased after 7 d. Conclusions:Adherence screening method and density gradient centrifugation can be effective methods to obtain BMSCs with high purity and rapid proliferation. Besides, the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.

  19. Treatment of APP/PS1 Double Transgenic Mice of Alzheimer ‘s Disease by Transplanting pEGFP/A2M (FP6) Transfected Neural Stem Cells into the Hippocampus%pEGFP/A2M(FP6)转染神经干细胞海马移植治疗APP/PS1双转基因AD小鼠的实验观察

    Institute of Scientific and Technical Information of China (English)

    武强; 廖光昊; 李露斯; 范文辉; 黎红华; 濮捷; 徐志鹏; 程鹤; 杨柳; 刘菲

    2013-01-01

    Objective: To observe the migration and differentiation of pEGFP/A2M (FP6) transfected neural stem cells (NSCs), the deposits of Aβ in the brain and the change of learning and memory ability of APP/PS1 double transgenic mice of Alzheimer's Disease (AD) after the NSCs were transplanted into the hippocampus. Methods: APP/PS1 transgenic AD mice were randomly divided into four groups: sham operated (SO) group, artificial cerebrospinal fluid (ACSF) group, transfected pEGFP neural stem cell (pEGFP-NSCs) group and transfected pEGFP/A2M(FP6) neural stem cell (pEGFP/A2M(FP6)-NSCs) group. The ACSF, pEGFP-NSCs or pEGFP/A2M (FP6)-NSCs were transplanted into the CA1 region of the hippocampus of the mice. The learning and memory ability of the mice were assessed with Mirror water maze test. The migration and differentiation of pEGFP/A2M(FP6) transfected NSCs and the deposits of Aβ in the brain of the mice were observed by immuno-histochemistry. Results: The latencies of pEGFP/A2M (FP6)-NSCs group and pEGFP-NSCs group were significantly shorter than that in the SO group and ASCF group (P<0.05). The latency of pEGFP/A2M(FP6)-NSCs group was shorter than that in the pEGFP-NSCs group (P<0.05). Anti-Aβ detection showed Aβ deposits in the hippocampus and cortex of pEGFP/A2M(FP6)-NSCs group and pEGFP-NSCs group were surrounded by transplanted NSCs. The amount and average size of Aβ deposits in the hippocampus and cortex of pEGFP/A2M (FP6)-NSCs group were reduced markedly, compared with the other three groups (P<0.05). The expression of Nestin was detecte after transplantation. Immunofluorescent detection indicated that majority of transplanted cells expressed GFAP while only a few cells expressed MAP-2. Conclusion: Transplantation of pEGFP/A2M (FP6)-NSCs into the hippocampal region of APP/PS1 double transgenic AD mice could reduce the Aβ deposits and promote the learning and memory ability. Partial transplanted NSCs will differentiate into neurons or astro-cytes.%目的:携带pEGFP

  20. Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury.

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    Shih-Tao Wen

    Full Text Available High concentrations of oxygen aggravate the severity of lung injury in patients requiring mechanical ventilation. Although mesenchymal stem cells have been shown to effectively attenuate various injured tissues, there is limited information regarding a role for amniotic fluid stem cells (AFSCs in treating acute lung injury. We hypothesized that intravenous delivery of AFSCs would attenuate lung injury in an experimental model of hyperoxia-induced lung injury. AFSCs were isolated from EGFP transgenic mice. The in vitro differentiation, surface markers, and migration of the AFSCs were assessed by specific staining, flow cytometry, and a co-culture system, respectively. The in vivo therapeutic potential of AFSCs was evaluated in a model of acute hyperoxia-induced lung injury in mice. The administration of AFSCs significantly reduced the hyperoxia-induced pulmonary inflammation, as reflected by significant reductions in lung wet/dry ratio, neutrophil counts, and the level of apoptosis, as well as reducing the levels of inflammatory cytokine (IL-1β, IL-6, and TNF-α and early-stage fibrosis in lung tissues. Moreover, EGFP-expressing AFSCs were detected and engrafted into a peripheral lung epithelial cell lineage by fluorescence microscopy and DAPI stain. Intravenous administration of AFSCs may offer a new therapeutic strategy for acute lung injury (ALI, for which efficient treatments are currently unavailable.

  1. Construction of recombinant plasmid pEGFP -N1 -NPRL2 and its effect on proliferation of nasopharyngeal carcinoma cells%pEGFP -N1-NPRL2真核表达载体的构建及其对人鼻咽癌细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    乔宇; 白荣平; 盛玉彪

    2016-01-01

    Objective:To construct the eukaryotic expression vector of pEGFP -N1 -NPRL2 and its expression in human nasopharyngeal carcinoma cell line CNE1.Methods:Total mRNA was extracted from human nasopharyngeal carcinoma CNE1 cells,NPRL2 gene was obtained by RT -PCR and cloned into pEGFP -N1 vector,then the recombi-nant pEGFP -N1 -NPRL2 plasmid was constructed and transfected into CNE1 cells by Lipofectamine 2000.The ex-pression of NPRL2 in CNE1 cells was detected by qRT -PCR and Western blot.Results:Corrected construction of pEGFP -N1 -NPRL2 was identified by double enzyme digestion and DNA sequencing.NPRL2 gene expressed by the transfected cells was testified by qRT -PCR and Western blot.Conclusion:The recombinant pEGFP -N1 -NPRL2 plasmid has been constructed successfully and can inhibit CNE1 cells proliferation in vitro.%目的:构建 pEGFP -N1-NPRL2真核表达载体并观察其对鼻咽癌细胞株 CNE1体外增殖的影响。方法:提取 CNE1细胞中总 RNA,RT -PCR 扩增 NPRL2并克隆至 pEGFP -N1载体,鉴定出阳性克隆送测序,以重组质粒转染 CNE1细胞。通过 Western blot 检测转染细胞中 NPRL2蛋白的表达,CCK -8法检测细胞增殖的变化。结果:成功构建了 pEGFP -N1-NPRL2真核表达载体,Western blot 法检测到 NPRL2蛋白的表达, CCK -8法检测发现 NPRL2能够明显抑制肿瘤细胞增殖(P <0.05)。结论:成功构建 pEGFP -N1-NPRL2真核表达载体并转染至 CNE1细胞,NPRL2可抑制肿瘤细胞的增殖。

  2. Surface display of monkey metallothionein {alpha} tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium

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    He, Xiaochuan; Chen, Wenli; Huang, Qiaoyun [Huazhong Agricultural Univ., Wuhan (China). State Key Lab. of Agricultural Microbiology

    2012-09-15

    Monkey metallothionein {alpha} domain tandem repeats (4mMT{alpha}), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC - 10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN-4mMT{alpha}-EGFP fusion, was constructed and used to target 4mMT{alpha} and EGFP on the surface of Pseudomonas putida X4 (CCTCC - 209319). The surface location of the INAXN-4mMT{alpha}-EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMT{alpha} on the engineered strains was four times higher than that of the wild-type X4. The Cd{sup 2+} accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu{sup 2+} and Zn{sup 2+}. Moreover, the surface-engineered strains could effectively bind Cd{sup 2+} under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMT{alpha}-EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa-EGFP. These results suggest that P. putida X4 expressing 4mMT{alpha}-EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants. (orig.)

  3. Act-1 core promoter region introduces high-performance transcription of EGFP gene expressed in Caenorhabditis elegans%Act-1核心启动子转录的EGFP基因在秀丽隐杆线虫体内的表达

    Institute of Scientific and Technical Information of China (English)

    周前进; 姜小磊; 张红丽; 杜爱芳

    2009-01-01

    克隆获得的秀丽隐杆线虫(Caenorhabditis elegans)Act-1基因的核心启动子,经BglⅡ和HindⅢ限制性内切酶消化后,与用相同酶消化的pEGFP-4.1载体连接(由pEGFP-N1去掉CMV启动子形成),构建重组表达载体Pact-EGFP.通过脂质体介导转染Veto细胞,结果发现EGFP在Vero细胞中有表达,但表达量很低.通过显微注射将Pact-EGFP与pRF4共注射到C. elegans性腺,结果发现EGFP能够在C. elegans的皮层、副皮层以及咽部表达,根据表达部位不同,获得了2种转基因线虫株.研究结果显示:EGFP在C. elegans体内的表达水平明显高于在Vero细胞内的表达,表明C.elegans Act-1基因的核心启动子区域可能存在与转录水平密切相关的独特的转录调节元件.该研究为进一步实现寄生性线虫基因在C.elegans表达提供了参考.

  4. Fusion Expression of Main Function Domain of env Gene of Avian Leukosis Virus Subgroup J and EGFP Gene in Sf9 Cells%J亚群禽白血病病毒env基因主要功能区和EGFP基因在Sf9细胞中的融合表达

    Institute of Scientific and Technical Information of China (English)

    李拓凡; 多婷; 梁雄燕; 顾玉芳; 杨玉莹

    2015-01-01

    为获得具备良好抗原性和生物活性的J亚群禽白血病病毒(AL V-J)env基因主要功能区的表达产物,采用特异性引物分别从保存的pMD 18T-envHB2010质粒、PIRES2-EGFP中扩增ALV-J env基因主要功能区和增强型绿色荧光蛋白(EGFP)基因,酶切、连接后插入pFastBac Daul载体,构建杆状病毒转移载体pFastBac Daul-env-EGFP,将其转化DH 10Bac感受态细胞制备重组杆粒Bacmid-env-EGFP,转染Sf9细胞进行真核表达.结果显示,转染了重组杆粒的Sf9细胞在倒置荧光显微镜下呈现亮绿荧光;以ALV-J单克隆抗体JE9进行Western blot检测,转染重组杆粒的Sf9细胞检测出约85 ku的条带.结果表明,目的基因在Sf9细胞中得到了良好的表达,为进一步研究ALV-J提供基础试验材料.

  5. Construction and identification of recombinant baculovirus vector to coexpress GDNF and EGFP gene%GDNF和EGFP双基因共表达重组杆状病毒载体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    陈艳春; 王俊; 王士礼; 蔡昌枰; 李彪; 张一帆; 郭睿

    2009-01-01

    目的 构建携带增强型绿色荧光蛋白(EGFP)和胶质细胞源性神经营养因子(GDNF)的重组杆状病毒载体.方法 将目的 基因(EGFP和GDNF)克隆人杆状病毒表达载体pFastBacDual中,构建重组质粒pFB-EGFP-GDNF并予酶切鉴定;将pFB-EGFP-GDNF转化到含杆状病毒穿梭载体Bacmid的DH10Bac感受态菌中,获得重组杆状病毒载体Bacmid-EGFP-GDNF,抽提质粒并行PCR鉴定;脂质体转染法将Bacmid-EGFP-GDNF转染Sf9细胞包装病毒;免疫荧光法检测Sf9细胞EGFP和GDNF蛋白表达.结果 目的 基因片段正确插入pFastBacDual载体中;重组Bacmid正确;Bacmid-EGFP-GDNF包装转染成功,获得较高病毒滴度;免疫荧光检测表明,Sf9细胞中GDNF和EGFP蛋白共表达.结论 成功构建重组杆状病毒Bacmid-EGFP-GDNF,转染SD细胞共表达GDNF和EGFP蛋白,为进一步研究GDNF蛋白对内耳的保护作用奠定了实验基础.%Objective To construct a novel enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) recombinant baculovirus. Methods The target gene(EGFP and GDNF) was cloned into baculovirus transfer vector pFastBacDual, pFB-EGFP-GDNF was constructed and restriction enzyme analysis was conducted. pFB-EGFP-GDNF was transposited with baculovirus shuttle vector (Bacmid) into DH10Bac competent cells, and recombination baculovirus vector Bacmid-EGFP-GDNF was constructed. The plasmid was extracted and PCR was performed for identification. Bacmid-EGFP-GDNF was transfected with Sf9 insect cell package virus by liposomal transfection method. Immunofluorescent staining was employed to detect the expression of EGFP and GDNF protein in St9 cells. Results The target gene fragment was correctly cloned into pFastBaeDual vector, and recombinant Bacmid was constructed. Bacmid-EGFP-GDNF was successfully transfected, and higher virus titer was obtained. The coexpression of GDNF and EGFP protein in Sf9 cells was identified by immunofluorescent staining

  6. Recombinant Plasmid ZLW/pEGFP-C2 Transfection into Schistosomula of Schistosoma japonicum%日本血吸虫表膜特异结合肽重组质粒ZLW/pEGFP-C2体外转染日本血吸虫童虫的研究

    Institute of Scientific and Technical Information of China (English)

    刘彦; 曾庆仁; 杨胜辉; 魏琦; 周军; 李立新; 兰玲梅

    2011-01-01

    microscope.At 48 hours after culture, total RNA and proteins from transfected schistosomula were extracted, and the presence of the transgenes (ZLW and EGFP) in schistosomula were confirmed by RT-PCR and Western blotting. At 24, 48, 72, and 96 hours after transfection, the schistosomula were counted by light microscope with methylene blue staining, pEGFP-C2 empty plasmid group and TBS group served as controls. Results The transfection rate was about 10%. The fluorescence of ZLW/EGFP protein was mainly localized in the tegument of the worms, especially abundant around oral sucker and ventral sucker. The expected size of 259 bp fragment was successfully amplified by RT-PCR and confirmed by DNA sequencing. Western blotting analysis showed that ZLW/EGFP was expressed in schistosomula. No statistically significant difference was established for schistosomula mortality among ZLW/pEGFP-C2 group (14.0%, 48.8% ), pEGFP-C2 group (15.9%, 45.7%) and TBS group (16.9%, 50.3%) at 24 and 48 hours after transfection (P>0.05). At 72 hours after transfection the mortality rate of ZLW/pEGFP-C2 group (92.7%) was significantly higher than that of pEGFP-C2 group (73.2%)(P<0.01), and after 96 h the mortality in ZLW/pEGFP-C2 group increased to 100%. Couclusion ZLW/pEGFP-C2 plasmid has been introduced into juvenile S. japonicum by immersion in 0.75% DMSO and high concentration of plasmid,and was expressed in the parasite.

  7. Thalidomide Effects in Patients with Hereditary Hemorrhagic Telangiectasia During Therapeutic Treatment and in Fli-EGFP Transgenic Zebrafish Model

    Institute of Scientific and Technical Information of China (English)

    Hong-Ling Peng; Yi-Fang Yi; Shun-Ke Zhou; Si-Si Xie; Guang-Sen Zhang

    2015-01-01

    Background: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by recurrent epistaxis,mucocutaneous telangiectasia, and arteriovenous malformations.The efficacy of traditional treatments for HHT is very limited.The aim of this study was to investigate the therapeutic role of thalidomide in HHT patients and the effect in FLI-EGFP transgenic zebrafish model.Methods: HHT was diagnosed according to Shovlin criteria.Five HHT patients were treated with thalidomide (100 mg/d).The Epistaxis Severity Score (ESS), telangiectasia spots, and hepatic computed tomography angiography (CTA) were used to assess the clinical efficacy of thalidomide.The Fli-EGFP zebrafish model was investigated for the effect of thalidomide on angiogenesis.Dynamic real-time polymerase chain reaction assay, ELISA and Western blotting from patient's peripheral blood mononuclear cells and plasma were used to detect the expression of transforming growth factor beta 3 (TGF-β3) messenger RNA (mRNA) and vascular endothelial growth factor (VEGF) protein before and after 6 months of thalidomide treatment.Results: The average ESS before and after thalidomide were 6.966 ± 3.093 and 1.799 ± 0.627, respectively (P =0.009).The "telangiectatic spot" on the tongue almost vanished;CTA examination of case 2 indicated a smaller proximal hepatic artery and decreased or ceased hepatic artery collateral circulation.The Fli-EGFP zebrafish model manifested discontinuous vessel development and vascular occlusion (7 of 10 fishes), and the TGF-β3 mRNA expression of five patients was lower after thalidomide therapy.The plasma VEGF protein expression was down-regulated in HHT patients.Conclusions: Thalidomide reverses telangiectasia and controls nosebleeds by down-regulating the expression of TGF-β3 and VEGF in HHT patients.It also leads to vascular remodeling in the zebrafish model.

  8. Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene.

    Science.gov (United States)

    Watanabe, Yoshihisa; Tsujimura, Atsushi; Aoki, Miku; Taguchi, Katsutoshi; Tanaka, Masaki

    2016-02-01

    The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.

  9. Construction of eukaryotic vector pEGFP-N1-IL-17 and screening of stably transfected pEGFP-N1-IL-17-U87MG cell%IL-17真核表达载体的构建及其在胶质瘤细胞株U87MG中的表达与筛选

    Institute of Scientific and Technical Information of China (English)

    胡锦辉; 邱潮林; 华玲; 谢秋华; 何小雪; 沈振华; 胡珺

    2013-01-01

    IL-17 has been demonstrated to promote tumor growth via certain pathways. In current studies IL-17 has been found to be highly expressed in glioma. This research aims to construct the eukaryotic vector pEGFP-Nl-IL-17 and makes it to be stably expressed in glioma cell line U87MG in order to provide the basis for studying its function in glioma progress. PBMC was collected from an idiopathic thrombocytopenic purpura (ITP) patient and the RNA was extracted. IL-17 cDNA was synthesized and cloned into PMD 19-T and pEGFP-N1 plasmid by SaI I and BamH enzymatic digestion. The clones were identified by enzymatic digestion and sequencing. The correct clone was amplified and plasmid DNA was then transfected into glioma cell line U87MG. pEGFP-N1-IL-17-U87MG cells that stably expressed IL-17 were obtained by limited dilution by G418 added. Correct clone was identified by fluorescence, qRTPCR and ELISA. The results showed that IL-17 cDNA、PMDR 19-T-IL-17, and pEGFP-N1-IL-17 were successfully obtained. And pEGFP-Nl-IL-17-U87MG cells that stably express IL-17 were identified through fluorescence, qRT-PCR and ELISA. MCP-1 mRNA was down regulated after U87MG cell was transfected by IL-17. Therefore the eukaryotic expression vector pEGFP-Nl-IL-17 has been successfully constructed and stably transfected into U87MG cell which could be used for study on IL-17's function in glioma tumorigenesis.%IL-17可以通过多种途径促进肿瘤发展,前期工作发现胶质瘤组织中IL-17高表达,本研究拟构建IL-17真核表达载体pEGFP-N1-IL-17,并且稳定转染胶质瘤细胞株U87MG,为研究IL-17在胶质瘤中作用提供基础.取血小板减少性紫癜自愿患者外周血2ml,Ficoll分离PBMC,提取RNA后经含BamH Ⅰ及Sal Ⅰ酶切位点引物逆转录成cDNA,连接T载体,酶切后与经相同酶切的载体pEGFP-N1连接,卡那霉素筛选,重组载体经Xfect试剂转染胶质瘤细胞U87MG,以G418筛选,单克隆阳性株扩大培养,经荧光、Real Time PCR及ELISA

  10. 不同方法转染人前列腺癌PC-3细胞pEGFP-N1基因的体外实验研究%Study of pEGFP-N1 transfection into human prostate cancer cell PC-3 by different transfecticion methods in vitro

    Institute of Scientific and Technical Information of China (English)

    吴作辉; 白文坤; 张吉臻; 张跃力; 申锷; 胡兵

    2012-01-01

    目的:探讨转染人前列腺癌PC-3细胞pEGFP-N1基因的最佳转染方法.方法:以超声微泡造影剂、超声辐照、脂质体转染及其相互结合的方法,将质粒pEGFP-N1基因转染人前列腺癌PC-3细胞,24 h后以荧光显微镜观察前列腺癌PC-3细胞中的绿色荧光蛋白表达情况,并用流式细胞仪测定转染率.结果:以超声+微泡+脂质体组基因转染效率最高,与其他组比较,差异均有统计学意义(P<0.05).结论:超声联合微泡与脂质体结合能明显提高pEGFP-N1基因在人前列腺癌细胞中的转染率,是一种较理想的基因转染方法.%Objective: To find a better method to transfer pEGFP-Nl into human PC-3 prostate cancer cell. Methods:Ultrasound contrast agent microbubbles, ultrasound, and lipofection method or combined with each other were used to transfer plasmid pEGFP into human prostate cancer PC-3 cells. The expression of pEGFP-Nl was studied by fluorescerce microscope and flow cytometry 24 hours after transfection. Results: Ultrasound combined with microbubble and liposome group had the best efficiency and had significant difference compared to other groups(P<0. 05). Conclusions:The use of ultrasound, in combination with microbubbles, could be a potential physical method for increasing liposome gene delivery efficiency.

  11. Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

    Directory of Open Access Journals (Sweden)

    Saurabh Singh

    2005-01-01

    Full Text Available During the early stages of embryogenesis, pluripotent neural crest cells (NCC are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR. The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

  12. Heterologous expression of rat testis GABAA receptor βt variant in Chinese hamster ovary cells

    Institute of Scientific and Technical Information of China (English)

    Shi-FengLi; Yu-GuangChen; Yuan-ChangYan; Yi-PingLi

    2004-01-01

    Aim: To study the characteristics and possible retention functionof specific sequence in the 5'-end of rat testis GABAA receptor β 3t variant, Methods: Rat testis GABAA receptor β 3t variant cDNA was cloned and inserted into two eukaryotic expression vectors of pEGFP-N1 and pEGFP-C1 respectively, which have EGFP reporter gene.

  13. Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Lars Boenicke; Bradley D. Howard; Ilka Vogel; Hoiger Kalthoff

    2003-01-01

    AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

  14. Noninvasive optical diagnostics of enhanced green fluorescent protein expression in skeletal muscle for comparison of electroporation and sonoporation efficiencies

    Science.gov (United States)

    Tamošiūnas, Mindaugas; Kadikis, Roberts; Saknīte, Inga; Baltušnikas, Juozas; Kilikevičius, Audrius; Lihachev, Alexey; Petrovska, Ramona; Jakovels, Dainis; Šatkauskas, Saulius

    2016-04-01

    We highlight the options available for noninvasive optical diagnostics of reporter gene expression in mouse tibialis cranialis muscle. An in vivo multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) expression, providing information on location and duration of EGFP expression and allowing quantification of EGFP expression levels. For EGFP coding plasmid (pEGFP-Nuc Vector, 10 μg/50 ml) transfection, we used electroporation or ultrasound enhanced microbubble cavitation [sonoporation (SP)]. The transcutaneous EGFP fluorescence in live mice was monitored over a period of one year using the described parameters: area of EGFP positive fibers, integral intensity, and mean intensity of EGFP fluorescence. The most efficient transfection of EGFP coding plasmid was achieved, when one high voltage and four low voltage electric pulses were applied. This protocol resulted in the highest short-term and long-term EGFP expression. Other electric pulse protocols as well as SP resulted in lower fluorescence intensities of EGFP in the transfected area. We conclude that noninvasive multispectral imaging technique combined with fluorescence spectroscopy point measurements is a suitable method to estimate the dynamics and efficiency of reporter gene transfection in vivo.

  15. Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice.

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    Miroslava Anderova

    Full Text Available Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4. As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K(+ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD or 50 mM K(+. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K(+, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K(+.

  16. Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

    Directory of Open Access Journals (Sweden)

    Kawakami Minoru

    2011-11-01

    Full Text Available Abstract Background Neural crest cells (NCCs are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs. Results In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA, we demonstrated that PDGF-AA acts as an NCC-attractant in embryos. We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine. Conclusions Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.

  17. Transgenic fluorescent zebrafish Tg(fli1:EGFP)y¹ for the identification of vasotoxicity within the zFET.

    Science.gov (United States)

    Delov, Vera; Muth-Köhne, Elke; Schäfers, Christoph; Fenske, Martina

    2014-05-01

    The fish embryo toxicity test (FET) is currently one of the most advocated animal alternative tests in ecotoxicology. To date, the application of the FET with zebrafish (zFET) has focused on acute toxicity assessment, where only lethal morphological effects are accounted for. An application of the zFET beyond acute toxicity, however, necessitates the establishment of more refined and quantifiable toxicological endpoints. A valuable tool in this context is the use of gene expression-dependent fluorescent markers that can even be measured in vivo. We investigated the application of embryos of Tg(fli1:EGFP)(y1) for the identification of vasotoxic substances within the zFET. Tg(fli1:EGFP)(y1) fish express enhanced GFP in the entire vasculature under the control of the fli1 promoter, and thus enable the visualization of vascular defects in live zebrafish embryos. We assessed the fli1 driven EGFP-expression in the intersegmental blood vessels (ISVs) qualitatively and quantitatively, and found an exposure concentration related increase in vascular damage for chemicals like triclosan, cartap and genistein. The fluorescence endpoint ISV-length allowed an earlier and more sensitive detection of vasotoxins than the bright field assessment method. In combination with the standard bright field morphological effect assessment, an increase in significance and value of the zFET for a mechanism-specific toxicity evaluation was achieved. This study highlights the benefits of using transgenic zebrafish as convenient tools for identifying toxicity in vivo and to increase sensitivity and specificity of the zFET.

  18. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  19. Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce.

    Science.gov (United States)

    Takeuchi, K; Frank, J F

    2001-03-01

    Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria. The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively. E. coli O157:H7 retained pEGFP during frozen storage at -80 degrees C. The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells. A low percentage of EGFP-expressing cells was nonviable. The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration. The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured. The application of EGFP as a marker for E. coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy. EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types. The numbers of E. coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato. E. coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces. EGFP can serve as a marker to characterize E. coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.

  20. Construction of pEGFP-N1-TGF-β1 recombinant plasmid and transfected it into primary cultured AEC-Ⅱ of neonatal piglet using lipofectamine 2000%建立pEGFP-N1-TGF-β1重组质粒和脂质体介导转染原代培养新生猪AEC-Ⅱ方法

    Institute of Scientific and Technical Information of China (English)

    徐艳; 孙波

    2016-01-01

    Objective To construct pEGFP-N1-TGF-β1 recombinant plasmid and transfect it into primary cultured neonatal piglet type Ⅱ alveolar epithelium cell (AEC-Ⅱ) by using lipofectamine 2000,in order to provide basis of methodology for producing recombinant plasmids for transplantation of transfected AEC-Ⅱ into ALI/ARDS animal model lungs.Methods PCR primers were designed to amplify the human TGF-β1 cDNA fragment from plasmid.XhoI and EcoRI were used for double digesting the empty plasmid pEGFP-N1 and cDNA fragment of human TGF-β1.Then the products of double enzyme digestion by using T4 DNA ligase were connected and transformed into DH5α and cultured over night for 16 hours.The structure of recombinant plasmid was identified by using PCR and base sequencing to verify the correctness of pEGFP-N1-TGF-β1 recombinant plasmid.It was then transfected into primarily cultured AEC-Ⅱ by lipofectamine2000 mediated transfection and cultured for another 48 hour.Plasmid DNA (pEGFP-N1-TGF-β1 recombinant plasmid) and lipofectamine 2000 were added into serum-free DMEM respectively,then DNA suspension and Lipofectamine 2000 suspension were blended together and added into cells.After 24-48 hours later,the expression level of enhanced green fluorescent protein (EGFP) was evaluated under fluorescence microscope.Results The structure of vector was verified as pEGFP-N1-TGF-β1 recombinant plasmid by using PCR and base sequencing.Green fluorescence found in some cells showed that the pEGFP-N1-TGF-β1 recombinant plasmids had been successfully transfected into primary cultured AEC-Ⅱ,however,the transfection efficiency still need tobe further improved such as repeating the transfection procedure once again or using adenovirus mediated transfection method to improve the efficiceny and to transplant the cells into animal lungs eventually.Conclusions pEGFP-N1-TGF-β1 recombinant plasmid was successfully constructed and,for the first time,transfected into primarily cultured AEC-Ⅱ of

  1. Neurogenic Differentiation of Murine Adipose Derived Stem Cells Transfected with EGFP in vitro

    Institute of Scientific and Technical Information of China (English)

    方忠; 杨琴; 熊伟; 李光辉; 肖骏; 郭风劲; 李锋; 陈安民

    2010-01-01

    Some studies indicate that adipose derived stem cells(ADSCs)can differentiate into adipogenic,chondrogenic,myogenic,and osteogenic cells in vitro.However,whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated.In this study,the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene,and then the cells were induced for neural differentiation.The morphology of those ADSCs began to change within two days which developed i...

  2. Enhanced Antitumor Activity of EGFP-EGF1-Conjugated Nanoparticles by a Multitargeting Strategy.

    Science.gov (United States)

    Zhang, Bo; Jiang, Ting; Ling, Li; Cao, Zhonglian; Zhao, Jingjing; Tuo, Yanyan; She, Xiaojian; Shen, Shun; Jiang, Xinguo; Hu, Yu; Pang, Zhiqing

    2016-04-13

    Tumor stromal cells have been increasingly recognized to interact with tumor parenchyma cells and promote tumor growth. Therefore, we speculated that therapeutics delivery to both parenchyma cells and stromal cells simultaneously might treat a tumor more effectively. Tissue factor (TF) was shown to be extensively located in a tumor and was abundantly sited in both tumor parenchyma cells and stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages, indicating it might function as a favorable target for drug delivery to multiple cell types simultaneously. EGFP-EGF1 is a fusion protein derived from factor VII, the natural ligand of TF. It retains the specific TF binding capability but does not cause coagulation. In the present study, a nanoparticle modified with EGFP-EGF1 (ENP) was constructed as a multitargeting drug delivery system. The protein binding experiment showed EGFP-EGF1 could bind well to A549 tumor cells and other stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages. Compared with unmodified nanoparticles (NP), ENP uptake by A549 cells and those stromal cells was significantly enhanced but inhibited by excessive free EGFP-EGF1. In addition, ENP induced more A549 tumor cell apoptosis than Taxol and NP when paclitaxel (PTX) was loaded. In vivo, ENP accumulated more specially in TF-overexpressed A549 tumors by in vivo imaging, mainly regions unoccupied by factor VII and targeted tumor parenchyma cells as well as different types of stromal cells by immunofluorescence staining. Treatment with PTX-loaded ENP (ENP-PTX) significantly reduced the A549 tumor growth in nude mice while NP-PTX- and Taxol-treated mice had lower response to the therapy. Furthermore, H&E and TUNEL staining revealed that ENP-PTX induced more severe tumor necrosis and more extensive cell apoptosis. Altogether, the present study demonstrated that ENP could target multiple key cell types

  3. Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro

    Science.gov (United States)

    Bastos, Matheus Silva e; de Souza, Luciana Ângelo; Onofre, Thiago Souza; Silva, Abelardo; de Almeida, Márcia Rogéria; Bressan, Gustavo Costa; Fietto, Juliana Lopes Rangel

    2017-01-01

    BACKGROUND Gene reporter-fluorescent cells have emerged as alternative method for drug screening. OBJECTIVE Achievement of constitutive expression of fluorescent protein GFP by Leishmania braziliensis as alternative method for drug screening. METHODS L. braziliensis-GFP was generated using Leishmania tarentolae pLEXSY-egfp for constitutive expression of GFP. Fluorescent cells were selected and subjected to standardisation tests of anti-promastigote and anti-intracellular amastigote assays. FINDINGS Our results showed that L. braziliensis-GFP method is faster and more sensitive than Allamar Blue-resazurin. MAIN CONCLUSION Transfected parasites maintained stable fluorescence after successive in vitro passages and pLEXSY system can be used to achieve non-L. tarentolae fluorescent cells. PMID:28177050

  4. Asymmetric localization of Numb:EGFP in dividing neuroepithelial cells during neurulation in Danio rerio.

    Science.gov (United States)

    Reugels, Alexander M; Boggetti, Barbara; Scheer, Nico; Campos-Ortega, José A

    2006-04-01

    In the neural plate and tube of the zebrafish embryo, cells divide with their mitotic spindles oriented parallel to the plane of the neuroepithelium, whilst in the neural keel and rod, the spindle is oriented perpendicular to it. This change is achieved by a 90 degrees rotation of the mitotic spindle. We cloned zebrafish homologues of the gene for the Drosophila cell fate determinant Numb, and analyzed the localization of EGFP fusion proteins in vivo in dividing neuroepithelial cells during neurulation. Whereas Numb isoform 3 and the related protein Numblike are localized in the cytoplasm, Numb isoform 1 is localized to the cell membrane. Time-lapse analyses showed that Numb 1 is distributed uniformly around the cell cortex in dividing cells during plate and keel stages, but becomes localized at the basolateral membrane of some dividing cells during the transition from neural rod to tube. Using in vitro mutagenesis and Numb:EGFP deletion constructs, we showed that the first 196 amino acids of Numb are sufficient for this localization. Furthermore, we found that an 11-amino acid insertion in the PTB domain is essential for localization to the cortex, whereas amino acids 2-12 mediate the basolateral localization in the neural tube stage.

  5. Construction of pEGFP-ChEgTrp as DNA model for multi-epitope vaccine against Echinococcus granulosus

    Directory of Open Access Journals (Sweden)

    M. Ahmadzadeh

    2015-12-01

    Full Text Available Background: Infection with Echinococcus granulosus causes hydatidosis in human and ruminants. With regards to the high prevalence of hydatidosis in Iran, dealing with this disease is important in terms of public health. Objective: The aim of this study was to construct pEGFP-ChEgTrp as DNA model for multi-epitope vaccine against Echinococcus granulosus Methods: This experimental study was conducted in the Razi Vaccine & Serum Research Institute, Karaj in 2013. Initially, epitopes stimulating the host immune response were predicted by IEDB Database and the coding sequences were made. The sequences were amplified by PCR. The PCR products were cloned into pEGFP-N1 vector after digestion with XhoI restriction enzyme. The bacteria containing recombinant plasmid were evaluated using Colony PCR, agarose gel electrophoresis and sequencing methods. Findings: Four peptides with 10 linear epitopes were predicted in EgTrp antigen. The nucleotide sequence coding ChEgTrp was amplified by PCR using specific primers and a 270 bp fragment was obtained. This fragment was cloned into pEGFP-N1 vector and the recombinant plasmid was confirmed by Colony PCR and agarose gel electrophoresis. For final confirmation, the recombinant plasmid was sequenced and the pEGFP-ChEgTrp was constructed. Conclusion: The ChEgTrp was successfully cloned into the pEGFP-N1 vector and this plasmid can be used to design DNA vaccines.

  6. [Research on construction of sheep lung adenomas virus pEGFP-C1/exJSRV-env and induction of malignant transformation in NIH3T3].

    Science.gov (United States)

    Zhang, Yu-Fei; Liu, Yue; Wang, Zhuan-Jia; Sun, Xiao-Lin; Liu, Shu-Ying

    2014-05-01

    This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.

  7. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles.

    Science.gov (United States)

    Gilbert, Leona; Toivola, Jouni; Välilehto, Outi; Saloniemi, Taija; Cunningham, Claire; White, Daniel; Mäkelä, Anna R; Korhonen, Eila; Vuento, Matti; Oker-Blom, Christian

    2006-12-08

    Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

  8. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

    Directory of Open Access Journals (Sweden)

    Vuento Matti

    2006-12-01

    Full Text Available Abstract Fluorescence correlation spectroscopy (FCS monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids were fused to the C-terminus of the enhanced green fluorescent protein (EGFP. The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs. The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

  9. [Preparation of a novel AAV-ITR gene expression mini vector in Sf9 insect cells via baculovirus].

    Science.gov (United States)

    Li, Taiming; Pan, Junjie; Qi, Jing; Zhang, Chun

    2015-08-01

    AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.

  10. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    Science.gov (United States)

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  11. 超声微泡介导pEGFP-N1转染大鼠牙囊细胞:细胞生物学性质相对稳定%pEGFP-N1 transfection of rat dental follicle cells under ultrasound-mediated lipid microbubble:transfected cells have a relatively stable biological property

    Institute of Scientific and Technical Information of China (English)

    冉玲; 李晓倩; 蒋欣益; 邓锋; 宋锦璘; 曹礼

    2014-01-01

    BACKGROUND:With the interaction of ultrasound and microbubbles, cavitation and mechanical effects undermine the integrity of the cel membrane, resulting in temporary and reversible holes, increasing the permeability of cel membranes, enhancing gene transfer and improving gene transfection efficiency. OBJECTIVE: To investigate the efficiency and safety of rat dental folicle cels transfected with pEGFP-N1 plasmid mediated by microbubble under ultrasonic irradiation. METHODS:The primary dental folicle cels from newborn rats were cultured in vitro and passaged to the 4th generation. Under different conditions, pEGFP-N1 was used to transfect rat dental folicle cels. By combining the ultrasonic intensity (0.5, 1 W/cm2) with the irradiation time (15, 30, 45, 60 seconds), we got the best conditions of ultrasonic irradiation for the next experiment. There were five groups: plasmid, microbubble+plasmid, ultrasound+plasmid, ultrasound+microbubble+plasmid, and liposomes+plasmid groups. The expression of pEGFP was observed by inverted fluorescence microscope 48 hours after transfection, and meanwhile, the proliferation inhibition rate of rat dental folicle cels was determined by MTT method. RESULTS AND CONCLUSION:Under the 0.5 W/cm2 ultrasound for 30 seconds, the transfection efficiency was obviously higher than that under the other combinations. Under the above-mentioned condition, the transfection efficiency of rat dental folicle cels with pEGFP-N1 plasmid was higher than that mediated by the traditional liposome, and the cel viability had no obvious changes. Under suitable conditions, ultrasound microbubble technology can safely and effecitively mediate the transfection of rat dental folicle cels with pEGFP-N1 plasmid, and transfected cels also have a stable biological property as normal dental folicle cels. Therefore, ultrasound microbubble technology can provide an ideal method of gene transfection in periodontal tissue engineering.%背景:利用超声波和微泡对比

  12. Generation of transgenic dogs that conditionally express green fluorescent protein.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  13. Adipose-derived stem cells transfected withpEGFP-OSX enhance bone formation during distraction osteogenesis%pEGFP-OSX修饰的脂肪干细胞促进牵张成骨中新骨形成

    Institute of Scientific and Technical Information of China (English)

    Qing-guo LAI; Shao-long SUN; Xiao-hong ZHOU; Chen-ping ZHANG; Kui-feng YUAN; Zhong-jun YANG; Sheng-lei LUO; Xiao-peng TANG; Jiang-bo CI

    2014-01-01

    This study was designed to investigate the effects of local delivery of adipose-derived stem cells (ADSCs) transfected with transcription factor osterix (OSX) on bone formation during distraction osteogenesis. New Zealand white rabbits (n=54) were randomly divided into three groups (18 rabbits per group). A directed cloning technique was used for the construction of recombinant plasmidpEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the right mandible of all experimental rabbits, rabbits in group A were treated with ADSCs transfected withpEGFP-OSX, group B with ADSCs transfected withpEGFP-N1, and group C with physio-logical saline. Radiographic and histological examinations were processed after half of the animals within each group were humanely kiled by injection of sodium pentothal at Week 2 or 6 after surgery. The distraction bone density was measured as its projectional bone mineral density (BMD). Three parameters were measured, namely, the thickness of new trabeculae (TNT), and the volumes of the newly generated cortical bone (NBV1) and the cancelous bone (NBV2) of the distracted regions. Good bone generation in the distraction areas was found in group A, which had the highest BMD, TNT, and NBV in the distraction zones among the groups. There was no significant difference in bone genera-tion in the distraction areas between groups B and C. The results indicate that the transplantation of ADSCs trans-fected withpEGFP-OSX can effectively promote bone generation during distractionin vivo.%研究目的:观察牵张间隙植入pEGFP-OSX修饰的脂肪干细胞对牵张成骨中新骨形成的作用。  创新要点:提出来源广泛容易获取的脂肪干细胞作为种子细胞,被基因 OSX修饰后,在体内环境下可能促进成骨。  研究方法:通过X射线检查、组织学检查等手段观察成骨效果,进行统计学分析。  重要结论:通过短期的动物试验发现,pEGFP

  14. Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Lai Zhiyong; Qin Qin; Yu Baofeng; Xie Jun; Gao Ranpeng; Zhang Tiantian; Li Chunfeng

    2014-01-01

    Background Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate,which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases.The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication,hence leading to cell apoptosis.At present,HSVtk gene usually acts as suicide gene to kill tumor cells.The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the α-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.Methods pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed.HL-7702 liver cells,HUH-7 HCC,and HepG2 HCC were transfected with the recombinant plasmids.HSVtK gene expression was detected using Western blotting analysis.HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent.The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.Results Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully.HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells.After G418 selection,a HepG2 cells line stably expressing HSVtk gene was acquired.With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P <0.05).Conclusion The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

  15. Construction of EGFP-labeling system for visualizing the infection process of Xanthomonas axonopodis pv. citri in planta.

    Science.gov (United States)

    Liu, Li-Ping; Deng, Zi-Niu; Qu, Jin-Wang; Yan, Jia-Wen; Catara, Vittoria; Li, Da-Zhi; Long, Gui-You; Li, Na

    2012-09-01

    Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.

  16. Gene transfer to hepatocellular carcinoma: transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors.

    Science.gov (United States)

    Gerolami, R; Uch, R; Jordier, F; Chapel, S; Bagnis, C; Bréchot, C; Mannoni, P

    2000-09-01

    Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5'-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

  17. TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm.

    Directory of Open Access Journals (Sweden)

    Nicole A J Krentz

    Full Text Available Human embryonic stem cells (hESCs have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-Associated protein (Cas to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.

  18. 脂质体转染胚盘细胞将外源质粒pEGFP-C2导入鹌鹑胚的研究%Introduction of foreign plasmid pEGFP-C2 into the quail embryo via liposome transfection blastoderm cells

    Institute of Scientific and Technical Information of China (English)

    李艳; 李洪涛; 段丹丹; 赵宗胜

    2013-01-01

    In order to identify the availability of liposome transfection of foreign DNA as an effective way in making transgenic quail,foreign plasmid pEGFP-C2 was injected into the subgerminal cavity of the fresh fertilized eggs before incubation,and the embryos of 6 day-old were collected to identify the distribution of the exogenous DNA, The positive ratio was 84%, the exogenous gene was expressed in embryos. Using G0 generation quail tissue for DNA extraction and PCR analysis, and tissue sections for fluorescent detection,the successful expression of foreign gene in G0 generation quail tissue was confirmed. This research results indicates the method is effective to produce transgenic quail.%运用脂质体转染第Ⅹ期胚盘细胞的方法将外源质粒pEGFP C2经脂质体包裹后注射到鹌鹑种蛋Ⅹ期胚盘下腔,孵化种蛋.提取孵化6d的鹌鹑胚基因组DNA,进行PCR检测,嵌合体阳性率为84%,证实外源基因在胚胎中的表达.对孵化至出壳的G0代鹌鹑组织进行DNA提取并PCR分析,以及组织切片荧光检测,证实外源基因在G0代鹌鹑组织中成功表达.结果表明脂质体转染胚盘细胞是一种制备转基因鹌鹑行之有效的方法.

  19. A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Dinh, Phat X.; Panda, Debasis; Das, Phani B.; Das, Subash C.; Das, Anshuman [School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States); The Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States); Pattnaik, Asit K., E-mail: apattnaik2@unl.edu [School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States); The Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States)

    2012-10-25

    Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.

  20. 腺病毒通过不同径路导入豚鼠耳蜗后的实验观察%Distributions of cells infected by AD5-EGFP infused in different ways in guinea pig's cochlear

    Institute of Scientific and Technical Information of China (English)

    韩朝; 迟放鲁; 杨娟梅; 李雯; 沈云珍; 高文元

    2009-01-01

    目的:探讨经不同径路将腺病毒导入豚鼠耳蜗后,被感染耳蜗细胞的分布情况.方法:使用构建有EGFP指示基因的腺病毒分别通过圆窗入路进入外淋巴系统,耳蜗侧壁钻孔中阶入路进入内淋巴系统,耳蜗冷冻切片和组织铺片观察腺病毒感染耳蜗细胞的分布情况.结果:由圆窗入路进入外淋巴系统的腺病毒可以感染血管纹的Ⅰ型、Ⅳ型和Ⅴ型纤维细胞、螺旋唇上细胞、前庭膜细胞、Ronsensal孔内的螺旋神经元、前庭阶和鼓阶的上皮细胞,内外毛细胞和支持细胞不被感染;而耳蜗侧壁钻孔中阶入路进入内淋巴系统的腺病毒可以感染听器的支持细胞和血管纹缘细胞等.结论:腺病毒是一种有效的豚鼠耳蜗细胞转染载体,导入耳蜗后,可以将目的基因转染到耳蜗细胞内.通过不同径路将腺病毒导入外、内淋巴系统,耳蜗被感染的细胞范围不一致.仅注入外淋巴系统的腺病毒无法感染内淋巴系统内的细胞,只有将病毒导入内淋巴系统,耳蜗支持细胞才被感染.%Objective:To explore the distribution of cells infected by AD5-EGFP infused in different ways in the cochlea of guinea pig. Method: AD5-EGFP was infused into the endolymphatic system through a hole on the lateral wall of the scala media or into the extralymphatic system through the round window membrane respectively. The infected cochlear cells confirmed by expression of EGFP were examined on the whole mount or cryostat sections. Result: In the cochleae in which AD5-EGFP was infused into the extralymphatic system through the round window membrane, expression of EFGP could be found in the type ?, ? and ? fibrocyte of the stria vascularis, superlimbal cells of the spiral lip, cells in Ressenal membrane, spiral ganglion neurons in Rosenthal hole and cells lining the inner wall of scala vestibular and scala tympani, indicating that these cells were infected by adenovirus. None of the inner, outer

  1. Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

    Directory of Open Access Journals (Sweden)

    Behra Martine

    2012-01-01

    Full Text Available Abstract Background Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals, supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. Results In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. Conclusions We present a Tg(tnks1bp1:EGFP stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.

  2. Dynamic Expression of Lgr6 in the Developing and Mature Mouse Cochlea

    Directory of Open Access Journals (Sweden)

    Yanping eZhang

    2015-05-01

    Full Text Available The Wnt/β-catenin signaling pathway plays important roles in mammalian inner ear development. Lgr5, one of the downstream target genes of the Wnt/β-catenin signaling pathway, has been reported to be a marker for inner ear hair cell progenitors. Lgr6 shares approximately 50% sequence homology with Lgr5 and has been identified as a stem cell marker in several organs. However, the detailed expression profiles of Lgr6 have not yet been investigated in the mouse inner ear. Here, we first used Lgr6-EGFP-Ires-CreERT2 mice to examine the spatiotemporal expression of Lgr6 protein in the cochlear duct during embryonic and postnatal development. Lgr6-EGFP was first observed in one row of prosensory cells in the middle and basal turn at embryonic day 15.5 (E15.5. From E18.5 to postnatal day 3 (P3, the expression of Lgr6-EGFP was restricted to the inner pillar cells (IPCs. From P7 to P15, the Lgr6-EGFP expression level gradually decreased in the IPCs and gradually increased in the inner border cells (IBCs. At P20, Lgr6-EGFP was only expressed in the IBCs, and by P30 Lgr6-EGFP expression had completely disappeared. Next, we demonstrated that Wnt/β-catenin signaling is required to maintain the Lgr6-EGFP expression in vitro. Finally, we demonstrated that the Lgr6-EGFP-positive cells isolated by flow cytometry could differentiate into myosin 7a-positive hair cells after 10 days in-culture, and this suggests that the Lgr6-positive cells might serve as the hair cell progenitor cells in the cochlea.

  3. Simple and efficient vectors for retrofitting BACs and PACs with mammalian neoR and EGFP marker genes.

    Science.gov (United States)

    Kaname, T; Huxley, C

    2001-03-21

    Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) are widely used to investigate the functions of genes and genomes in mammalian cells in vitro and in vivo. We have developed a series of vectors which can simply and efficiently be retrofitted onto BACs or PACs. These vectors carry a neoR gene for selection in cells in tissue culture, including ES cells, and also an EGFP gene driven by the strong CAG promoter for quick detection of the DNA in cells. All the plasmids are retrofitted using the loxP site and Cre recombinase and some carry the gamma origin of plasmid R6K which does not function in commonly used bacteria such as DH10B. Retrofitting of PACs and BACs carrying alphoid DNA was very efficient with almost no rearrangement of the highly repetitive alphoid DNA. Following transfer into HT1080 cells and mouse oocytes in tissue culture the DNA could easily be monitored by the EGFP fluorescence.

  4. Hepatoma cell-specific ganciclovir-mediated toxicity of a lentivirally transduced HSV-TkEGFP fusion protein gene placed under the control of rat alpha-fetoprotein gene regulatory sequences.

    Science.gov (United States)

    Uch, Rathviro; Gérolami, René; Faivre, Jamila; Hardwigsen, Jean; Mathieu, Sylvie; Mannoni, Patrice; Bagnis, Claude

    2003-09-01

    Suicide gene therapy combining herpes simplex virus thymidine kinase gene transfer and ganciclovir administration can be envisioned as a powerful therapeutical approach in the treatment of hepatocellular carcinoma; however, safety issues regarding transgene expression in parenchyma cells have to be addressed. In this study, we constructed LATKW, a lentiviral vector expressing the HSV-TkEGFP gene placed under the control of the promoter elements that control the expression of the rat alpha-fetoprotein, and assayed its specific expression in vitro in hepatocarcinoma and nonhepatocarcinoma human cell lines, and in epidermal growth factor stimulated human primary hepatocytes. Using LATKW, a strong expression of the transgene was found in transduced hepatocarcinoma cells compared to a very low expression in nonhepatocarcinoma human cell lines, as assessed by Northern blot, RT-PCR, FACS analysis and ganciclovir-mediated toxicity assay, and no expression was found in lentivirally transduced normal human hepatocytes. Altogether, these results demonstrate the possibility to use a lentivirally transduced expression unit containing the rat alpha-fetoprotein promoter to restrict the HSV-TK-mediated induced GCV sensitivity to human hepatocarcinoma cells.

  5. Expression and Subcellular Localization of Recombinant SDF-1 and Its Mutant Intrakine in Transfected COS-7 Cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-yuan; GUO Zhi-gang; TAN-yi; MA Wei-feng; CAI Shao-xi; DU-jun; CAI Shao-hui

    2006-01-01

    Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-1/54 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein. This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface. Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-Dec/PET-30a(+)with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 /SDF-1/KDEL, pcDNA3.1 /SDF-1/54/KDEL, pEGFP/SDF-1/KDEL and pEGFP/SDF-1/54/KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate, they were transferred into COS-7 cells with liposome. The exogenous expressions were observed, fusion protein SDF-1/His and SDF-1/54/His were confirmed by Western blot, and the SDF-1/EGFP and SDF-1/54/EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1/KDEL/His and SDF-1/54 KDEL/His expressed in COS-7 cells. Subcelluar localization analysis showed that SDF-1/KDEL/EGFP and SDF-1/54/KDEL/EGFP were located mainly in endoplasmic reticulum. Conclusion: Four expression vectors pcDNA3. 1/ SDF -1/KDEL, pcDNA3.1/SDF - 1/54/KDEL, pEGFP/SDF - 1/KDEL and pEGFP/SDF-1/54/KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum.

  6. Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

    Science.gov (United States)

    Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

    2016-12-30

    This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

  7. In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

    Institute of Scientific and Technical Information of China (English)

    Jintao Li; Qi Yan; Xingbao Zhu; Dan Xu; Tinghua Wang; Huatang Zhang; Jia Liu

    2009-01-01

    BACKGROUND: Neurotrophin-4 (NT-4) can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its activity in the central nervous system. Delivering NT-4 into the central nervous system via cells or vectors may have therapeutic benefit.OBJECTIVE: To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-N1.DESIGN, TIME AND SETTING: Neural genetic engineering experiment. The study was performed at the Neuroscience Institute of Kunming Medical College between October 2007 and March 2008.MATERIALS: The pEGFP-N1 plasmid vector was provided by Kunming Institute of Zoology, Chinese Academy of Sciences; embryonic brain tissues were provided by the First Affiliated Hospital of Kunming Medical College. TRIzol RNA extraction Kit was purchased from Sigma (USA), One Step RNA PCR Kit (AMV) etc. were from Takara (Dalian, China).METHODS: Total RNA was extracted from human embryonic brain tissues using Trizol. The agarose gel electrophoresis showed two bands: 18 S and 28 S, which were essential subunits of total RNA. The human NT-4 DNA was obtained via RT-PCR and inserted into the pEGFP-N1 vector using ligation and transformation reaction.MAIN OUTCOME MEASURES: The sequencing results of the DNA in the recombinant of NT-4-pEGFP-N1.RESULTS: The NT-4-pEGFP-N1 vector was sequence-verified and showed the expected molecular weight.CONCLUSION: The recombinant of NT-4-pEGFP-N1 was constructed successfully in vitro.

  8. Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

    Science.gov (United States)

    Yee, Karen K; Li, Yan; Redding, Kevin M; Iwatsuki, Ken; Margolskee, Robert F; Jiang, Peihua

    2013-05-01

    Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue.

  9. Synthesis, characterization, and cytotoxicity of the plasmid EGFP-p53 loaded on pullulan-spermine magnetic nanoparticles

    Science.gov (United States)

    Eslaminejad, Touba; Nematollahi-Mahani, Seyed Noureddin; Ansari, Mehdi

    2016-03-01

    Magnetic nanoparticles have been used as effective vehicles for the targeted delivery of therapeutic agents that can be controlled in their concentration and distribution to a desired part of the body by using externally driven magnets. This study focuses on the synthesis, characterization, and functionalization of pullulan-spermine (PS) magnetic nanoparticles for medical applications. Magnetite nanopowder was produced by thermal decomposition of goethite (FeOOH) in oleic acid and 1-octadecene; pullulan-spermine was deposited on the magnetite nanoparticles in the form of pullulan-spermine clusters. EGFP-p53 plasmid was loaded on functionalized iron oleate to transfer into cells. Synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), vibrating sample magnetometry (VSM), and transmission electron microscopy (TEM). The encapsulation efficiency and drug loading efficiency of the nanocomplexes were tested. FTIR studies showed the presence of oleic acid and 1-octadecene in the iron oleate nanopowder and verified the interaction between spermine and pullulan. The characteristic bands of PS in the spectrum of the pullulan-spermine-coated iron oleate (PSCFO) confirmed that PS covered the surface of the iron oleate particles. TEM studies showed the average size of the iron oleate nanopowder, the PSCFO, and the plasmid-carrying PSCFO (PSCFO/pEGFP-p53) to be 34±12 nm, 100±50 nm and 172±3 nm, respectively. Magnetic measurements revealed that magnetic saturation of the PSCFO was lower in comparison with the iron oleate nanopowder due to the presence of organic compounds in the former. In cytotoxicity tests performed using U87 cells as glioblastoma cells, a 92% survival rate was observed at 50 μg/μl of the plasmid-carrying PSCFO, with an IC50 value of 189 μg/μl.

  10. Construction of recombinant plasmid pEGFP-N2-GPC3 and effects of GPC3 gene on the growth promotion of growth factors in GPC3-SK-Hep-1%重组质粒pEGFP-N2-GPC3的构建及GPC3对生长因子FGF2、IGF2促进细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    王冰; 林山; 王烈

    2008-01-01

    目的 构建GPC3基因真核表达重组质粒,观察GPC3对生长因子FGF2、IGF2促进肝癌细胞增殖的影响.方法 自行设计引物从GPC3原核扩增重组质粒pDNR-LIB.GPC3中获得编码GPC3的基因片段.应用基因重组技术,将GPC3基因片段克隆到真核表达载体pEGFP-N2中,然后经脂质体介导转染SK-Hep-1,并通过C418(600 mg/L)筛选出抗性克隆,应用荧光显微镜及逆转录-聚合酶链反应(RT-PER)法对转染细胞内pEGFP-GPC3的表达进行鉴定,采用噻唑蓝(MTT)比色法研究GPC3对生长因子FGF2、IGF2促细胞增殖效应的影响.结果 限制性内切酶酶切分析、重组质粒测序鉴定表明为正确重组子,荧光显微镜下可见转染的SK-Hep-1胞膜区发出强绿色荧光,RT-PCR表明GPC3在SK-Hep-1中成功表达,生长因子实验中,GPC3明显抑制FGF2促细胞增殖效应,与空质粒转染组比较差异有统计学意义(P0.05).结论 测序表明,编码的氨基酸序列与人GPC3完全一致;构建完成真核表达重组质粒pEGFP-N2-GPC3;GPC3基因在SK-Hep-1中成功表达;GPC3在FGF2信号通路中可能发挥负性调控因子的作用.%Objective To construct a eukaryotic expression recombinant plasmid named pEGFP-N2-GPC3, and study the effects of GPC3 gene on the growth promotion of growth factors in GPC3-SK-Hep-1. Methods Human GPC3 full-length eDNA was obtained from pDNR-LIB-GPC3 prokaryotic amplified recombinant plasmid by PCR. The target gene GPC3 was inserted into the eukaryotic expression vector pEGFP-N2. The recombinant plasmid was transfected into the SK-Hep-1 human hepatoma carcinoma cells via Lipofeetin transfection. Transfected SK-Hep-1 cells were selectively screened with G418 (600 mg/L).The expression of GPC3 gene in transfected SK-Hep-1 was detected by fluorescence microscopy and RT-PCR. Effects of GPC3 gene on the growth promotion of growth factors in GPC3-SK-Hep-1 were assayed by MTL Results The recombinant plasmid was identified to be right for ORF by

  11. 中国人常见GJB2基因突变表达载体的构建及鉴定%Construction of GJB2 mutations common in Chinese EGFP fusion protein vectors

    Institute of Scientific and Technical Information of China (English)

    张延平; 张元丁; 李丽娜; 马磊; 孙玉蕊; 张宗霖; 刘金伟; 邓惠严; 祝威

    2009-01-01

    Objective:To construct GJB2 gene mutaitons common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutaitons in GJB2 gene. Method: Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutaiton methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutaions were inserted into pEG-FP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were trans-fected with the recombinant DNA samples by the liposome complex method. Results The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence singals were distributed uniformly in cytoplasm. Conclusion; GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.%目的:构建中国人常见GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp与增强型绿色荧光蛋白(EGFP)的融合蛋白表达载体,寻找体外研究GJB2基因缺失突变致聋机制的有效途径.方法:用体外定点突变法构建235 de1C、299-300 delAT和176 de1 16 bp突变全序列与EGFP表达载体,以此为模板PCR扩增突变有效表达序列,将PCR产物连接到pMD19-T载体中,EcoRI/BamHI双酶切克隆载体,测序鉴定序列正确性后,将酶切产物插入pEGFP-N1载体中,脂质体转染HEK293细胞,荧光显微镜观察表达的融合蛋白.结果:GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp在HEK293细胞中高效表达,表达主要位于细胞质中.结论:成功构建了中国人常见GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp与EGFP的融合蛋白表达载体,为进一步研究其致聋机制奠定了基础.

  12. Synthesis, characterization, and cytotoxicity of the plasmid EGFP-p53 loaded on pullulan–spermine magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Eslaminejad, Touba, E-mail: tslaminejad@yahoo.com [Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Nematollahi-Mahani, Seyed Noureddin, E-mail: nnematollahi@kmu.ac.ir [Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Neuroscience Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Afzal Research Institute, Kerman (Iran, Islamic Republic of); Ansari, Mehdi, E-mail: mansari@kmu.ac.ir [Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Pharmaceutics Research Centre, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of)

    2016-03-15

    Magnetic nanoparticles have been used as effective vehicles for the targeted delivery of therapeutic agents that can be controlled in their concentration and distribution to a desired part of the body by using externally driven magnets. This study focuses on the synthesis, characterization, and functionalization of pullulan–spermine (PS) magnetic nanoparticles for medical applications. Magnetite nanopowder was produced by thermal decomposition of goethite (FeOOH) in oleic acid and 1-octadecene; pullulan–spermine was deposited on the magnetite nanoparticles in the form of pullulan–spermine clusters. EGFP-p53 plasmid was loaded on functionalized iron oleate to transfer into cells. Synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), vibrating sample magnetometry (VSM), and transmission electron microscopy (TEM). The encapsulation efficiency and drug loading efficiency of the nanocomplexes were tested. FTIR studies showed the presence of oleic acid and 1-octadecene in the iron oleate nanopowder and verified the interaction between spermine and pullulan. The characteristic bands of PS in the spectrum of the pullulan–spermine-coated iron oleate (PSCFO) confirmed that PS covered the surface of the iron oleate particles. TEM studies showed the average size of the iron oleate nanopowder, the PSCFO, and the plasmid-carrying PSCFO (PSCFO/pEGFP-p53) to be 34±12 nm, 100±50 nm and 172±3 nm, respectively. Magnetic measurements revealed that magnetic saturation of the PSCFO was lower in comparison with the iron oleate nanopowder due to the presence of organic compounds in the former. In cytotoxicity tests performed using U87 cells as glioblastoma cells, a 92% survival rate was observed at 50 µg/µl of the plasmid-carrying PSCFO, with an IC{sub 50} value of 189 µg/µl. - Highlights: • Magnetite nanopowder was produced by thermal decomposition method. • TEM studies showed the average size of

  13. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  14. Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

    Institute of Scientific and Technical Information of China (English)

    Yong-Nan Xu; Joon-Ho Yoon; Dae-Hwan Ko; Teoan Kim; Nam-Hyung Kim; Sang-Jun Uhm; Bon-Chul Koo; Mo-Sun Kwon; Ji-Yeol Roh; Jung-Seok Yang; Hyun-Yong Choi; Young-Tae Heo; Xiang-Shun Cui

    2013-01-01

    The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins.Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer,but these approaches have been largely ineffective; however,a third approach,lentivirus-mediated transgenesis,has successfully produced transgenic livestock.In this study,we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors,which expressed enhanced green fluorescent protein (EGFP) systemically.Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV)carrying EGFP were injected into the perivitelline space of MII oocytes.EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5% ± 2.2% v.s.22.9% ± 2.9%).Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers.Ten heifers were successfully impregnated and delivered 10 healthy calves.All of these calves expressed EGFP as detected by in vivo imaging,PCR and Southern blotting.In addition,we established an EGFP-expressing cell line from TG calves,which was followed by nuclear transfer (NT).Recloned 8-cell embryos also expressed EGFP,and there were no differences in the rates of fusion,cleavage and development between cells derived from TG and non-TG calves,which were subsequently used for NT.These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle.Moreover,our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.

  15. Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

    Science.gov (United States)

    Furness, John B; Cho, Hyun-Jung; Hunne, Billie; Hirayama, Haruko; Callaghan, Brid P; Lomax, Alan E; Brock, James A

    2012-06-01

    Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with

  16. Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

    Full Text Available We have constructed virus-like particles (VLPs harboring hemagglutinin (HA, neuraminidase (NA, matrix protein 1 (M1 ,and proton channel protein (M2 using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

  17. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  18. Metabolomic analysis of riboswitch containing E. coli recombinant expression system.

    Science.gov (United States)

    Muhamadali, Howbeer; Xu, Yun; Morra, Rosa; Trivedi, Drupad K; Rattray, Nicholas J W; Dixon, Neil; Goodacre, Royston

    2016-02-01

    In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli cells. This metabolic burden analysis was performed against a number of recombinant expression systems and control strains and included: (i) standard transcriptional recombinant expression control system BL21(DE3) with the expression plasmid pET-eGFP, (ii) the recently developed dual transcriptional-translational recombinant expression control strain BL21(IL3), with pET-eGFP, (iii) BL21(DE3) with an empty expression plasmid pET, (iv) BL21(IL3) with an empty expression plasmid, and (v) BL21(DE3) without an expression plasmid; all strains were cultured under various induction conditions. The growth profiles of all strains together with the results gathered by the analysis of the Fourier transform infrared (FT-IR) spectroscopy data, identified IPTG-dependent induction as the dominant factor hampering cellular growth and metabolism, which was in general agreement with the findings of GC-MS analysis of cell extracts and media samples. In addition, the exposure of host cells to the synthetic inducer ligand, pyrimido[4,5-d] pyrimidine-2,4-diamine (PPDA), of the orthogonal riboswitch containing expression system (BL21(IL3)) did not display any detrimental effects, and its detected levels in all the samples were at similar levels, emphasising the inability of the cells to metabolise PPDA. The overall results obtained in this study suggested that although the BL21(DE3)-EGFP and BL21(IL3)-EGFP strains produced comparable levels of recombinant eGFP, the presence of the orthogonal riboswitch seemed to be moderating the metabolic burden of eGFP production in the cells enabling higher biomass yield, whilst providing a greater level of control over protein expression.

  19. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  20. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  1. Diffusion behavior of the fluorescent proteins eGFP and Dreiklang in solvents of different viscosity monitored by fluorescence correlation spectroscopy

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-12-01

    Fluorescence correlation spectroscopy relies on temporal autocorrelation analysis of fluorescence intensity fluctuations that spontaneously arise in systems at equilibrium due to molecular motion and changes of state that cause changes in fluorescence, such as triplet state transition, photoisomerization and other photophysical transformations, to determine the rates of these processes. The stability of a fluorescent molecule against dark state conversion is of particular concern for chromophores intended to be used as reference tags for comparing diffusion processes on multiple time scales. In this work, we analyzed properties of two fluorescent proteins, the photoswitchable Dreiklang and its parental eGFP, in solvents of different viscosity to vary the diffusion time through the observation volume element by several orders of magnitude. In contrast to eGFP, Dreiklang undergoes a dark-state conversion on the time scale of tens to hundreds of microseconds under conditions of intense fluorescence excitation, which results in artificially shortened diffusion times if the diffusional motion through the observation volume is sufficiently slowed down. Such photophysical quenching processes have also been observed in FCS studies on other photoswitchable fluorescent proteins including Citrine, from which Dreiklang was derived by genetic engineering. This property readily explains the discrepancies observed previously between the diffusion times of eGFP- and Dreiklang-labeled plasma membrane protein complexes.

  2. Influence on behavior of rats with Parkinson's disease treated by bone mesenchymal stem cell modified by plasmid pIRESneo-EGFP-BDNF%转染pIRESneo-EGFP-BDNF的骨髓间充质干细胞侧脑室注射对帕金森大鼠行为学的影响

    Institute of Scientific and Technical Information of China (English)

    张平; 赵钢勇; 宋月平; 苏立凯

    2012-01-01

    [目的]观察转染pIRESneo- EGFP-BDNF的骨髓间充质干细胞(MSCs)侧脑室注射对帕金森病(PD)大鼠行为学的影响.[方法]将pEGFP(N1)-BDNF与pIRESneo进行双酶切后再连接,构建高拷贝质粒pIRESneo-EGFPBDNF,采用电穿孔法将其转染MSCs.采用6-羟多巴(6-OHDA)制备PD大鼠模型(36只),造模成功后将其随机分为3组,每组12只.B组为模型组:侧脑室注射生理盐水;C组:侧脑室注射MSCs;D组:侧脑室注射转染pIRESneoEGFP-BDNF的MSCs;A组(12只大鼠,为假手术组):以生理盐水代替6-OHDA后,侧脑室注射生理盐水.分别于侧脑室注射后2、4、8周腹腔注射阿扑吗啡(APO)诱导大鼠旋转,观察各组大鼠行为学变化.[结果]经双酶切鉴定,pIRESneo-EGFP-BDNF构建成功.侧脑室注射细胞干预PD大鼠模型后2、4、8周,大鼠旋转次数D组<C组<B组(P均<0.05);D组大鼠旋转次数明显减少,但仍较A组多.[结论]侧脑室注射转染plRESneo-EGFP-BDNF的MSCs能明显改善PD大鼠的行为能力.%Objective To observe influence on behavior of Parkinson's disease( PD) model treated by bone mesen-chymal stem cells ( MSCs) modified by plasmid pIRESneo-EGFP-BDNF. Methods pEGFP-BDNF and pIRESneo were double enzymed and then were reconstructed into pIRESneo-EGFP-BDNF which was transfected to MSCs with electroproa-lion. 36 rat models of PD were set up by 6-OHDA and divided into three groups randomly; group B( group model): inlrac-erebrovenlricular injection of saline; group C: intracerebroventricular injection of bone MSCs; group D: intracerebroventric-ular injection of bone MSCs modefied by pIRESneo-ECFP-BDNF. Group A (12 rats, sham opreation groups): saline instead of 6-OHDA, than intracerebroventricular injection of saline. The rotating behavior of rat models induced by Apomor-phine intraperitoneally which transplanting bone MSCs or MSCs modified by plasmid pIRESneo-EGFP-BDNF through cerebral lateral ventricle after 2, 4 and 8 weeks. Results Pbsmids pIRESneo-EGFP

  3. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    Science.gov (United States)

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application.

  4. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  5. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian;

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon......-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear...

  6. Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

    Institute of Scientific and Technical Information of China (English)

    FENG Yong; ZHENG Dong; YANG Shuhua; LI Jing

    2007-01-01

    The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR Ⅰ, Pst Ⅰ and bound to eukaryotic expression plasmid pIRES2-EGFP to construct eukaryotic expression plasmid pIRES2-EGFP-bFGF. The pIRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supematants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the pIRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type Ⅰ and Ⅲ collagen mRNA, suggesting that pIRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.

  7. The Construction of the EGFP Enterovirus 71%肠道病毒71型(EV71)荧光病毒的构建

    Institute of Scientific and Technical Information of China (English)

    朱守海; 王璐璐

    2014-01-01

    人肠道病毒71型(Enterovirus,EV71)是引起手足口病的主要病原体。首先利用反向遗传学的方法,构建了EV71全长cDNA感染性克隆,经体外转录、转染RD细胞后成功获得了拯救病毒。随后,将增强型绿色荧光蛋白基因(Enhanced green fluorescent protein,EGFP)插入到EV71基因组中构建荧光病毒。结果表明,此荧光病毒不仅可以侵染、复制,在传代的过程中EGFP基因也保持了较好的稳定性,表明其可以作为一种报告病毒应用于高通量的EV71抗病毒药物筛选中。%Human Enterovirus 71(EV71)is one of the major agents causing hand, foot and mouth disease(HFMD). An infectious full-length cDNA clone of EV71 as well as the recuse virus were obtained using the reverse genetic techniques. Then, the EGFP gene was inserted into the EV71 genome to construct the reporter virus. The results show that the EGFP-EV71 virus not only are replication-competent and infectious, but the EGFP gene are stable during passaging, implicating the recombinant virus could be used as a reporter virus for high throughput EV71 antiviral drugs screening.

  8. Global expression profiling of globose basal cells and neurogenic progression within the olfactory epithelium.

    Science.gov (United States)

    Krolewski, Richard C; Packard, Adam; Schwob, James E

    2013-03-01

    Ongoing, lifelong neurogenesis maintains the neuronal population of the olfactory epithelium in the face of piecemeal neuronal turnover and restores it following wholesale loss. The molecular phenotypes corresponding to different stages along the progression from multipotent globose basal cell (GBC) progenitor to differentiated olfactory sensory neuron are poorly characterized. We used the transgenic expression of enhanced green fluorescent protein (eGFP) and cell surface markers to FACS-isolate ΔSox2-eGFP(+) GBCs, Neurog1-eGFP(+) GBCs and immature neurons, and ΔOMP-eGFP(+) mature neurons from normal adult mice. In addition, the latter two populations were also collected 3 weeks after olfactory bulb ablation, a lesion that results in persistently elevated neurogenesis. Global profiling of mRNA from the populations indicates that all stages of neurogenesis share a cohort of >2,100 genes that are upregulated compared to sustentacular cells. A further cohort of >1,200 genes are specifically upregulated in GBCs as compared to sustentacular cells and differentiated neurons. The increased rate of neurogenesis caused by olfactory bulbectomy had little effect on the transcriptional profile of the Neurog1-eGFP(+) population. In contrast, the abbreviated lifespan of ΔOMP-eGFP(+) neurons born in the absence of the bulb correlated with substantial differences in gene expression as compared to the mature neurons of the normal epithelium. Detailed examination of the specific genes upregulated in the different progenitor populations revealed that the chromatin modifying complex proteins LSD1 and coREST were expressed sequentially in upstream ΔSox2-eGFP(+) GBCs and Neurog1-eGFP(+) GBCs/immature neurons. The expression patterns of these proteins are dynamically regulated after activation of the epithelium by methyl bromide lesion.

  9. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K

    2011-01-01

    To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A...... domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (¿40) or 25-aa (¿25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. ¿40 conferred efficient growth characteristics...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...

  10. Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

    Institute of Scientific and Technical Information of China (English)

    郑风荣; 孙修勤; 刘洪展; 吴兴安; 钟楠; 王波; 周国栋

    2010-01-01

    Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic losses.In this study,we aimed to develop the DNA vaccine against LCDV,using DNA vaccination technology.We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate.The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line.The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also ana...

  11. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence.

    Directory of Open Access Journals (Sweden)

    Yu-Ju Lin

    Full Text Available Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.

  12. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence.

    Science.gov (United States)

    Lin, Yu-Ju; Huang, Li-Hsin; Huang, Ching-Tsan

    2013-01-01

    Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.

  13. Establishment of HEK293 cell line expressing GFPAQP1 to determine water osmotic permeability

    Institute of Scientific and Technical Information of China (English)

    Jun-weiGAO; He-mingYU; Qian-liuSONG; Shu-xinLI; Xue-junLI

    2004-01-01

    AIM: To develop an osmotic water permeability assay.METHODS: We subcloned the rat AQP1 cDNA into pEGFPC3 vector. HEK293 cells were transfected with pEGFP-C3/AQP 1 or pEGFP-C3 and selected by G418. The expression of AQP1was detected by RT-PCR and Western blot. Confocal laser fluorescence microscopy was used to record the change of fluorescent density corresponding to the volume change of the cells

  14. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L;

    1999-01-01

    of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  15. Establishment of a Tumor-bearing Mouse Model Stably Expressing Human Tumor Antigens Survivin and MUC1 VNTRs

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li-xing; DU Jian-shi; WANG Yu-qian; LIU Chen-lu; XIA Qiu; ZHANG Xi-zhen; CONG Xian-ling; ZHANG Hai-hong

    2012-01-01

    The eukaryotic vectors VR1012 expressing survivin or 33 tandem repeats of human mucin 1(MUC1)(VNTRs),namely,VR1012-S and VR1012-VNTR(VNTR=variable number of tandem repeat),were constructed by cloning survivin and VNTR genes into VR1012,respectively.The eukaryotic vector pEGFP expressing survivin and MUC1 VNTRs fusion gene pEGFP-MS was also constructed.Mouse melanoma cell line(B16)stably expressing survivin and MUC1 VNTRs(MS+B16)was established by Lipofectamine-mediated transfection of pEGFP-MS into B16 cells.EGFP expression in MS+B16 cells was observed using a fluorescent microscope and survivin and MUC1 VNTRs(MS)expression was confirmed by means of Western blot analysis.A syngenic graft tumor model was generated by subcutaneous injection of MS+B16 cells into C57/BL6 mice and tumor size increased rapidly with time in a cell number dependent manner.After the third immunization,mice were challenged subcutaneously with 5×105 MS+B16 cells.Compared with that of the negative control immunized with phosphate-buffered saline(PBS),a significant reduction of tumor growth was observed in groups immunized with survivin plasmid DNA and MUC1 VNTRs plasmid DNA.Thus,the suppression of subcutaneous tumor was antigen-specific.This model is useful for the development of tumor vaccines targeting survivin and MUCI VNTRs.

  16. Molecular and functional characterization of GAD67-expressing, newborn granule cells in mouse dentate gyrus

    Science.gov (United States)

    Cabezas, Carolina; Irinopoulou, Theano; Cauli, Bruno; Poncer, Jean Christophe

    2013-01-01

    Dentate gyrus granule cells (GCs) have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2–4 weeks post-mitotic GCs. These rather immature cells are able to fire action potentials (APs) and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus GCs is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage. PMID:23565079

  17. Cloning and expression and preliminary study of the pathogenic mechanism of SAll rotavirus VP3 gene%SA11株轮状病毒VP3基因的克隆表达及致病机制的初步研究

    Institute of Scientific and Technical Information of China (English)

    李明; 何海洋; 徐广振; 尹怡; 薛毅; 吴玉章; 李晋涛

    2011-01-01

    To explore the relationship between SAllVP3 and the host cell, as well as study its potential molecular mechanism, the co-expression vector of VP3 of SAll with EGFP was constructed. RT-PCR was preformed to amplify VP3 gene fragments from the reverse transcription product of RV genome RNA. The VP3 gene fragments were harvested and inserted into pEGFP-C1 toconstruct the pEGFP-C1/VP3 expression vector. And the expression in 293T cell was observed under a fluorescence microscope and analyzed by Western blotting. As a control with pEGFP-C1/Rb94 plasmid, the inhibition of VP3 protein to GFP gene was detected by Flow cytometry,fluorescence microscope and Western blotting. Western blotting demonstrated that pEGFP-C1/VP3 could express the VP3 protein in 293T cell. Fluorescence intensity of pEGFP-C1/VP3 group was significant weaker than that of pEGFP-C1/Rb94 group. In conclusion, a recombinant eukaryotic co-expression vector of pEGFP-C1/VP3 was successfully constructed, which is effectively expressed in eukaryotic cells. VP3 protein may inhibit the expression of host cell gene. All the results provide a possible pathogenic mechanism of rotavirus VP3.%目的 克隆、真核表达SAll株轮状病毒VP3基因,对其致病机制进行了初步研究.方法 用RT-PCR从SAll株总RNA中扩增出VP3基因,并克隆到真核表达载体pEGFP-C1上,构建重组表达载体pEGFP-C1/VP3,用荧光显微镜及Western blot检测VP3基因在真核细胞(293T细胞)的表达情况.以重组质粒pEGFP-C1/Rb94为对照,用流式、荧光显微镜、Westernblot观察VP3蛋白对GFP基因表达的抑制作用.结果 在293T细胞中检测到VP3基因的表达;pEGFP-C1/VP3组的流式荧光强度、Western blot的蛋白条带大小均明显弱于pEGFP-C1/Rb94对照组.结论 成功构建了pEGFP-C1/VP3真核穿梭表达载体,在真核细胞中有效表达,实验结果 提示VP3蛋白可能对宿主细胞大分子蛋白的表达有抑制作用,可能是轮状病毒的一种新的致病机制.

  18. Development and characterization of a clinical strain of Coxsackievirus A16 and an eGFP infectious clone

    Institute of Scientific and Technical Information of China (English)

    Chenglin; Deng; Xiaodan; Li; Siqing; Liu; Linlin; Xu; Hanqing; Ye; Cheng-Feng; Qin; Bo; Zhang

    2015-01-01

    Coxsackievirus A16(CA16) is one of the major causes of hand, foot, and mouth disease(HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious public health problem in Asia. Therefore, it is important to understand the pathogenesis and replication of CA16. In this study, a stable infectious c DNA clone of an epidemic strain of Coxsackievirus A16(CA16) was assembled, and subsequently a reporter virus(e GFP-CA16) was constructed by inserting the e GFP gene between the 5’-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site(ITTLG) at its C-terminus. This was transfected into Vero cells to generate infectious recombinant viruses. The growth characteristics and plaque morphology, in vitro, in mammalian cells were found to be indistinguishable between the parental and recombinant viruses. Although the e GFP-CA16 showed smaller plaque size as compared to recombinant CA16, both were found to exhibit similar growth trends and EC50 of NITD008. In summary, this stable infectious c DNA clone should provide a valuable experimental system to study CA16 infection and host response. The e GFP-CA16 is expected to provide a powerful tool to monitor e GFP expression in infected cells and to evaluate the antiviral activity of potential antiviral agents in the treatment of CA16 infections.

  19. Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

    Directory of Open Access Journals (Sweden)

    Zeinab Soleimanifar

    2016-10-01

    Full Text Available Background: Luteinizing hormone (LH was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly.

  20. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

    Science.gov (United States)

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes. PMID:28216629

  1. EXPRESSION OF A MUTANT hTERT IN HUMAN BLADDER CARCINOMA CELL LINE T24 AND ITS CLINICAL SIGNIFICANCE

    Institute of Scientific and Technical Information of China (English)

    符伟军; 洪宝发; 黄君健; 徐兵; 高江平; 王晓雄; 黄翠芬

    2004-01-01

    Objective: To construct a mutant pEGFP- hTERT expression vector, to observe its steady expression in transfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods: PCR amplification was performed by using primers based on the known gene sequence of hTERT. PCR production was cloned into plasmid pGEMT-T easy and the sequence of mutant hTERT gene was analyzed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. After transfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes of bladder carcinoma cell line T24 were detected by light microscopy and senescence correlated β-galactosidase staining. Results: Identification of pEGFP-hTERT by enzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated β-galactosidase in transfected cells gradually increased with extended cultured time and cell growth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.

  2. Liposome enhanced transfection of pEGFP-N1 plasmid to prostate cancer cells in vitro with low frequency ultrasound combined with microbubbles%低频超声联合微泡造影剂增强脂质体介导pEGFP-N1基因转染体外前列腺癌细胞

    Institute of Scientific and Technical Information of China (English)

    吴作辉; 白文坤; 张吉臻; 胡兵

    2012-01-01

    目的 探讨低频超声联合微泡造影剂增强脂质体介导增强型绿色荧光蛋白质粒(pEGFP-N1)转染前列腺癌细胞的可行性,并对超声微泡浓度参数进行优化.方法 将前列腺癌PC-3细胞悬液分为空白对照组、超声组、微泡组、微泡+超声组、脂质体组、脂质体+微泡组、脂质体+超声组、脂质体+微泡+超声组,其中脂质体+微泡+超声组根据微泡体积浓度不同分为(0、10%、20%、30%、40%和50%)6个亚组.经超声辐照,24 h后用荧光显微镜观察细胞中基因表达情况,并用流式细胞仪检测转染率.结果 脂质体+微泡+超声组基因转染效率最高,与其他组比较差异均有统计学意义( P均<0.05);在脂质体+微泡+超声亚组中,微泡浓度为20%亚组基因转染率最高.结论 低频超声联合微泡能有效增强脂质体介导pEGFP-N1基因在体外前列腺癌细胞中的转染率,20%是体外基因转染前列腺癌细胞的最佳微泡浓度.%Objective To investigate the feasibility of liposome enhanced transfection of green fluorescent protein gene (pEGFP-N1) plasmid to prostate cancer cells with low frequency ultrasound combined with microbubbles, and to optimize the parameters of microbubbles concentration. Methods PC-3 prostate cancer cell suspension was divided into 8 groups > i. e. control group, ultrasound group, microbubbles group, microbubbles+ultrasound group, liposome group, microbubbles + liposome group, liposome+ultrasound group and ultrasound + microbubbles + liposome group. The ultrasound-f-micro-bubbles+liposome group was classified into 6 sub-groups: 0, 10% , 20% , 30% , 40% , and 50% , based on microbubbles volume concentration. The cell suspension was cultured in 12-well plates for 24 h after irradiation, and fluorescent microscopy was used to observe gene transfection and calculated the rate of gene transfection. Results Ultrasound+microbubbles + liposome group had the best efficiency, which was

  3. The specific expression mediated by promoters of hepatitis B virus in hepatocarcinoma cells%肝部细胞中乙肝病毒启动子介导的特异性表达

    Institute of Scientific and Technical Information of China (English)

    谢娜; 王晓燕; 张琼

    2006-01-01

    Objective: To investigate four expression vectors carrying enhanced green fluorescent protein (EGFP) genes,which under the control of different HBV promoters,were detected and compared as to their expressions in hepatocytes and nonhepatic cell lines.Methods: Four HBV promoters (including enhancers) were amplified by PCR,subcloned into the expression vector pEGFP-1.The four recombinants controlled by HBV promoters were analyzed and identified by restriction enzymes and sequencing.After transfection into human hepatoma cell lines and non-liver cells,transfection efficiency was measured by EGFP expression through fluorescence microscopy and analyzed with fluorescence activated cell sorter (FACS).Results: All target fragments were separately obtained and successfully cloned into the expression vector.The transfection results showed that in hepatoma cells,the expression of EGFP was more obvious than it was in non-hepatic cells.Among the four promoters,S gene promoter presented the strongest transfection efficiency.Conclusion: Our findings indicate that HBV promoters could lead specific expression in hepatocytes,different promoters had different outcomes,which might be a potential for the gene therapy of liver diseases.

  4. Colonization of Azospirillum brasilense Yu62 in Wheat Via EGFP%巴西固氮螺菌Yu62的EGFP标记及其在小麦体内的定殖研究

    Institute of Scientific and Technical Information of China (English)

    刘华伟; 王庆贺; 张宏; 王蕊; 肖红利; 郭蔼光

    2009-01-01

    以质粒pEGFP-C1为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因全长序列,将其与原核表达载体pVK-100连接,构建成重组载体pVK-EGFP.利用电转化法将重组载体导入巴西固氮螺菌Yu62中,得到EGFP标记菌株.用EGFP标记菌接种小麦'小偃107'种子,室内限菌条件下培养10 d后,用荧光显微镜观测标记菌在小麦体内的定殖规律并观察接菌植株的田间生长状况.结果显示,巴西固氮螺菌Yu62能定殖于小麦根毛区、茎组织的细胞间隙等部位,而且接菌小麦'小偃107'植株在根系发育、株高、分蘖数等方面比对照有较明显的优势.研究表明,巴西固氮螺菌Yu62能够定殖于小麦根茎内,并具有促进植物生长的作用.

  5. Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Ying Wang; Hui-Xiong Xu; Ming-De Lu; Qing Tang

    2008-01-01

    AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript 11 KDR-TK plasmid by enzymatic digestion with XhoI and Sa/I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble,pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP)expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTr) assay.RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to the prodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively.CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs.Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

  6. Stable Somatic Gene Expression in Mouse Lungs Following Electroporation-mediated Tol2 Transposon Delivery.

    Science.gov (United States)

    Muliawan, Hary Sakti; Nakayama, Kazuhiko; Yagi, Keiko; Ikeda, Koji; Yagita, Kazuhiro; Hirata, Ken-ichi; Emoto, Noriaki

    2015-10-07

    Gene delivery to the lung has rapidly progressed as an important method for studying various chronic lung diseases. Viral vectors, albeit highly efficient, are limited by the host immune response. Electroporation, a well-known non-viral method, can efficiently deliver genes to the lung, but is unable to induce stable gene expression. The Tol2 transposon is another non-viral method that can induce stable gene expression by reinserting its genes into the host genome. In this study, we combined electroporation and Tol2 transposons to obtain stable, high-level gene expression in the mouse lung. Tol2 transposon plasmids (pT2A-EGFP; Tol2, pCAGGS-TP; transposase) were optimized in vitro, and the electroporation procedure (pCAG-EGFP) was optimized in mouse lungs. After optimization, a combination of electroporation plus the Tol2 transposon was used in a comparative analysis with electroporation plus pCAG-EGFP. GFP expression levels were quantified and visualized on days 4 and 7 post-electroporation. We successfully reproduced the Tol2 transposon system in vitro and the electroporation procedure in vivo. We observed sustainable GFP expression using electroporation plus the Tol2 transposon on days 4 and 7, while electroporation plus pCAG-EGFP resulted in decreased GFP expression on day 7. We were able to induce high-level, stable gene expression in mouse lungs using a combination of electroporation and the Tol2 transposon. This represents a safer method for lung gene delivery that can be used as an alternative to viral vectors.

  7. Validation of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein, for the screening of estrogenic activity in calf urine

    NARCIS (Netherlands)

    Bovee, T.F.H.; Heskamp, H.H.; Hamers, A.R.M.; Hoogenboom, L.A.P.; Nielen, M.W.F.

    2005-01-01

    Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor a (hERa) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, this yeast estrogen assay was validated as a qualitative screening

  8. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds on both receptor types

    NARCIS (Netherlands)

    Bovee, T.F.H.; Helsdingen, J.R.; Rietjens, I.M.C.M.; Keijer, J.; Hoogenboom, L.A.P.

    2004-01-01

    Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor (hER) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic

  9. Morphology, input-output relations and synaptic connectivity of Cajal-Retzius cells in layer 1 of the developing neocortex of CXCR4-EGFP mice.

    Science.gov (United States)

    Anstötz, Max; Cosgrove, Kathleen E; Hack, Iris; Mugnaini, Enrico; Maccaferri, Gianmaria; Lübke, Joachim H R

    2014-11-01

    Layer 1 (L1) neurons, in particular Cajal-Retzius (CR) cells are among the earliest generated neurons in the neocortex. However, their role and that of L1 GABAergic interneurons in the establishment of an early cortical microcircuit are still poorly understood. Thus, the morphology of whole-cell recorded and biocytin-filled CR cells was investigated in postnatal day (P) 7-11 old CXCR4-EGFP mice where CR cells can be easily identified by their fluorescent appearance. Confocal-, light- and subsequent electron microscopy was performed to investigate their developmental regulation, morphology, synaptic input-output relationships and electrophysiological properties. CR cells reached their peak in occurrence between P4 to P7 and from thereon declined to almost complete disappearance at P14 by undergoing selective cell death through apoptosis. CR cells formed a dense and long-range horizontal network in layer 1 with a remarkable high density of synaptic boutons along their axons. They received dense GABAergic and non-GABAergic synaptic input and in turn provided synaptic output preferentially with spines or shafts of terminal tuft dendrites of pyramidal neurons. Interestingly, no dye-coupling between CR cells with other cortical neurons was observed as reported for other species, however, biocytin-labeling of individual CR cells leads to co-staining of L1 end foot astrocytes. Electrophysiologically, CR cells are characterized by a high input resistance and a characteristic firing pattern. Increasing depolarizing currents lead to action potential of decreasing amplitude and increasing half width, often terminated by a depolarization block. The presence of membrane excitability, the high density of CR cells in layer 1, their long-range horizontal axonal projection together with a high density of synaptic boutons and their synaptic input-output relationship suggest that they are an integral part of an early cortical network important not only in layer 1 but also for the

  10. T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp3

    Directory of Open Access Journals (Sweden)

    Quatromoni Jon G

    2011-11-01

    Full Text Available Abstract Regulatory T cells (Treg that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg or induced Treg (iTreg converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL and splenocytes (SPL in B16 murine melanoma-bearing C57BL/6 Foxp3EGFP mice. OT-II Foxp3EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA. Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR and exposure to transforming growth factor β (TGFβ. B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment.

  11. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes.

    Science.gov (United States)

    Zhu, Wuyang; Li, Jiangjiao; Tang, Li; Wang, Huanqin; Li, Jia; Fu, Juanjuan; Liang, Guodong

    2011-07-10

    To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (G.luc) were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  12. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  13. The study on apoptosis of liver cancer cell line HepG2 induced by culture supernatant of DCs transfection with recombinant plasmid pEGFP-N1/CpG-HBcAg(ISS)%重组质粒pEGFP-N1/CpG-HBcAg(ISS)转染DC培养上清诱导HepG2株凋亡的研究

    Institute of Scientific and Technical Information of China (English)

    周健; 田德英; 许东; 张振纲; 陈淼; 章述军; 吴会玲

    2010-01-01

    目的:探讨人类乙肝核心抗原重组质粒pEGFP-N1/CpG-HBcAg(ISS)转染人外周血单核细胞来源树突状细胞后,细胞培养上清诱导肝癌细胞株HepG2凋亡的作用及机制.方法:构建真核表达质粒pEGFP-N1/CpG-HBcAg(ISSa,c),将其转染人外周血来源DC,用培养上清诱导HepG2的凋亡.用流式细胞仪检测已转染DC表面CD80和CD86的表达,检测培养上清诱导HepG2凋亡的变化.用ELISA法检测转染后DC培养上清的IFN-γ、IL-2、IL-12、IL-4和IL-10的水平.结果:pEGFP-N1/CpG-HBcAg(ISSa)转染DC表面CD80和CD86的表达均有明显升高(P<0.01).转染后上清中Th1型细胞因子 IFN-γ、IL-2和IL-12的表达增强(P<0.01),Th2型细胞因子IL-4和IL-10的表达下降(P<0.05),pEGFP-N1/CpG-HBcAg(ISSa)组培养上清对HepG2细胞具有促凋亡作用,随着培养时间延长,细胞凋亡率逐渐增加,HepG2细胞在诱导后24小时凋亡率达到最大,为18.4%.结论:重组质粒pEGFP-N1/CpG-HBcAg(ISSa)转染培养上清能明显促进肝癌细胞株HepG2的凋亡.

  14. Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein)genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

  15. Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum

    OpenAIRE

    Ibáñez-Sandoval, Osvaldo; Tecuapetla, Fatuel; Unal, Bengi; Shah, Fulva; Koós, Tibor; Tepper, James M.

    2010-01-01

    Whole-cell recordings were obtained from tyrosine hydroxylase-expressing (TH+) neurons in striatal slices from bacterial artificial chromosome transgenic mice that synthesize enhanced green fluorescent protein (EGFP) selectively in neurons expressing TH transcriptional regulatory sequences. Stereological cell counting indicated that there were ~2700 EGFP–TH+ neurons/striatum. Whole-cell recordings in striatal slices demonstrated that EGFP–TH+ neurons comprise four electrophysiologically disti...

  16. Expression of a model gene in prostate cancer cells lentivirally transduced in vitro and in vivo.

    Science.gov (United States)

    Bastide, C; Maroc, N; Bladou, F; Hassoun, J; Maitland, N; Mannoni, P; Bagnis, C

    2003-01-01

    In a preclinical model for prostate cancer gene therapy, we have tested lentiviral vectors as a practical possibility for the transfer and long-term expression of the EGFP gene both in vitro and in vivo. The human prostate cancer cell lines DU145 and PC3 were transduced using experimental conditions which permitted analysis of the expression from a single proviral vector per cell. The transduced cells stably expressed the EGFP transgene for 4 months. After injection of the transduced cell populations into Nod-SCID mice a decrease in EGFP was only observed in a minority of cases, while the majority of tumors maintained transgene expression at in vitro levels. In vivo injection of viral vector preparations directly into pre-established subcutaneous or orthotopic tumor masses, obtained by implantation of untransduced PC3 and DU145 cells led to a high transduction efficiency. While the efficiency of direct intratumoral transduction was proportional to the dose of virus injected, the results indicated some technical limitations inherent in these approaches to prostate cancer gene therapy.

  17. 聚乙烯亚胺介导BmkCT基因抑制C6胶质瘤细胞C-Myc、VEGF表达%Expression of Polyethyleneimine-mediated Bmk CT Gene Inhibitation to VEGF and C-Myc in C6 Glioma Cells

    Institute of Scientific and Technical Information of China (English)

    张亚涛; 范丽娜; 温春丽; 胡风云

    2016-01-01

    目的:应用阳离子聚合物聚乙烯亚胺(polyethyleneimine,PEI)介导东亚钳蝎氯离子通道毒素( Buthus martensii Karsch Chlorotoxin-like Toxin,Bmk CT)基因转染 C6胶质瘤细胞,观察其对 C6细胞内 C-Myc、VEGF 表达的影响。方法:将 PEI 分别和 pEGFP-N1-Bmk CT、pEGFP-N1质粒混合,获得 PEI / pEGFP-N1-Bmk CT 和 PEI / pEG-FP-N1两种复合物,将其转染 C6细胞,MTS 法观察细胞增殖和存活活力,48 h后通过 Western-blot 方法检测 C-Myc、VEGF 蛋白表达水平。结果:PEI / pEGFP-N1-Bmk CT 转染 C6细胞48 h后较 PEI / pEGFP-N1能够显著抑制C6细胞增殖和存活活力,同时抑制 C-Myc、VEGF 蛋白表达。结论:PEI / pEGFP-N1-Bmk CT 转染 C6细胞可能通过抑制 C-Myc、VEGF 表达进而抑制 C6细胞增殖和血管生成。%Objective:To investigate the effects of PEI(polyethyleneimine)-mediated Bmk CT(Buthus martensii Karsch Chlorotoxin-like Toxin),Bmk CT gene transfection on the expression levels of C-Myc and VEGF of C6 glioma cells. Methods:pEGFP-N1 and pEGFP-N1-BmK CT were combined with PEI to form the composites,PEI/ pEGFP-N1-Bmk CT and PEI/ pEGFP-N1,which were used to transfect C6 glioma cells. Then,MTS test was performed to investigate the proliferation and viability of the transfected cells. After 48 hours the expression of C-Myc and VEGF proteins were de-tected by Western-bloting. Results:The proliferation of C6 glioma cells in the pEGFP-N1-Bmk CT group was significant-ly inhibited,while the expression levels of C-Myc and VEGF were lower than pEGFP-N1 group after the transfection. Conclusion:PEI/ pEGFP-N1-Bmk CT may suppress the angiogenesis and the proliferation of C6 glioma cells through in-hibiting the expression of C-Myc and VEGF.

  18. The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola.

    Directory of Open Access Journals (Sweden)

    Scott A C Godfrey

    2011-03-01

    Full Text Available Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1, which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1, revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.

  19. Mitochondrial physiology and gene expression analyses reveal metabolic and translational dysregulation in oocyte-induced somatic nuclear reprogramming.

    Directory of Open Access Journals (Sweden)

    Telma C Esteves

    Full Text Available While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT, the enucleated oocyte (ooplasm must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene. We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism.

  20. Characterization of gene expression regulated by human OTK18 using Drosophila melanogaster as a model system for innate immunity

    Indian Academy of Sciences (India)

    Cole R. Spresser; Sarah E. Marshall; Kimberly A. Carlson

    2008-08-01

    OTK18 is a human transcriptional suppressor implicated in the regulation of human immunodeficiency virus type-one infection of mononuclear phagocytes. It is ubiquitously expressed in all normal tissues, but its normal homeostatic function is yet to be characterized. One hypothesis is that OTK18 aids in the regulation of the innate immune system. To test this hypothesis, cDNA microarray analysis was performed on the total RNA extracted from Drosophila melanogaster embryonic Schneider 2 (S2) cells transfected with either pEGFP-OTK18 (enhanced green fluorescent protein) or empty vector controls (pEGFP-N3) for 6, 12 and 24 h. cDNA microarray analysis revealed differential expression of genes known to be important in regulation of Drosophila innate immunity. The expression levels of two genes, Metchnikowin and CG16708 were verified by quantitative real-time reverse transcription PCR. These results suggest a role for OTK18 in innate immunity.

  1. Eukaryotic Expression of Swamp Buffalo Follistatin%沼泽型水牛卵泡抑素基因的真核表达研究

    Institute of Scientific and Technical Information of China (English)

    邓继贤; 杨秀荣; 韦英明; 蒋和生

    2011-01-01

    In this study,follistatin gene was chosen as the candidate gene affecting the reproductive ability in buffalo. Re-combinant plasmid pEGFP-bFS was conducted and expressed in both buffalo fetal fibroblast (BFF) and BHK cells. pMD-bFS and pEGFP-Ni were digested with Xho Ⅰ ,Sac Ⅱ ,then eukaryotic expression vector-pEGFP-bFS was constructed. Buffalo fetal fibroblast and BHK cells were cultured with pEGFP-bFS intermixing LipofectamineTM 2000 in carbon dioxide incubator with 37 ℃ ,5% CO2 ,100% humidity. After 24 h,expression levels of recombinant vector were verified through observing directly under phase contrast fluorescence microscope, RT-PCR and Western blotting. The results indicated that bFST expressed in both buffalo fibroblast and BHK cells. This study was a very significance to produce bioactive bFS engineered plasmid and research effects of bFS on reproductive ability.%试验通过候选基因法,选定卵泡抑素(follistatin,FS)作为影响水牛繁殖性能的主要候选基因,通过基因工程的方法,用Xho Ⅰ、Sac Ⅱ双酶切广西大学动物遗传育种与繁殖实验室克隆的添加有ACC的Kozaka序列的pMD-bFS质粒和pEGFP-N1,构建pEGFP-bFS真核表达质粒,将其与阳离子脂质体混匀后,分别转染体外培养的水牛胎儿成纤维(BFF)细胞和仓鼠肾(BHK)细胞系,经过48 h培养后,在相差荧光显微镜下观察绿色荧光蛋白的表达水平,用RT-PCR和Western blotting方法对转入质粒表达进行定性鉴定.结果显示,本研究成功构建了添加有ACC的Kozaka序列的pEGFP-bFS真核表达质粒,该重组质粒在BFF和HBK两种细胞均表达,但在HBK细胞系的表达量稍高.本研究结果将为下一步制备具有生物活性的bFS工程疫苗,研究bFS对水牛繁殖性能的影响提供参考.

  2. Efficient generation of human embryonic stem cell-derived cardiac progenitors based on tissue-specific enhanced green fluorescence protein expression.

    Science.gov (United States)

    Szebényi, Kornélia; Péntek, Adrienn; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I; Sarkadi, Balázs; Apáti, Ágota

    2015-01-01

    Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFP(high) rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFP(high) rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFP(high) rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications.

  3. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals

    Science.gov (United States)

    Sim, Chan Kyu; Kim, Inki

    2016-01-01

    Interleukin-7 (IL-7) is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP) gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease. PMID:27589392

  4. Stable transgene expression in primitive human CD34+ hematopoietic stem/progenitor cells, using the Sleeping Beauty transposon system.

    Science.gov (United States)

    Sumiyoshi, Teiko; Holt, Nathalia G; Hollis, Roger P; Ge, Shundi; Cannon, Paula M; Crooks, Gay M; Kohn, Donald B

    2009-12-01

    Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell

  5. Construction and Co-expression of Bicistronic Plasmid Encoding Human WEE1 and Stem Cell Factor

    Institute of Scientific and Technical Information of China (English)

    Ping LEI; Wen-Han LI; Wen-Jun LIAO; Bing YU; Hui-Fen ZHU; Jing-Fang SHAO; Guan-Xin SHEN

    2005-01-01

    To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy.In the present study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and enhanced green fluorescent protein (EGFP) separately with internal ribosome entry site (IRES). We first examined the expression and location of WEE1Hu in Chinese hamster ovary (CHO) cells and showed that WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells,which were proved by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry,respectively. Furthermore, inhibition of cisplatin-induced apoptosis was observed in CD34+ cells transfected with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our study suggests that the introduction of two functional genes via bicistronic vector is more powerful and efficient than single gene therapy.

  6. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  7. Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene

    Institute of Scientific and Technical Information of China (English)

    HUANG Tao; JIANG Xiao-dan; XU Zhong; YUAN Jun; DING Lian-shu; ZOU Yu-xi; XU Ru-xiang

    2005-01-01

    Objective: To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene. Methods: Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR. Results: The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2. Conclusions: The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.

  8. Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression.

    Science.gov (United States)

    Fernandes, Fabiana; Vidigal, João; Dias, Mafalda M; Prather, Kristala L J; Coroadinha, Ana S; Teixeira, Ana P; Alves, Paula M

    2012-11-01

    Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research.

  9. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  10. Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Kim, Min Sun; Kim, Ki Hong

    2012-03-01

    To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-ΔNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-ΔNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-ΔNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-ΔNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-ΔNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish.

  11. Apical localization of ASIP/PAR-3:EGFP in zebrafish neuroepithelial cells involves the oligomerization domain CR1, the PDZ domains, and the C-terminal portion of the protein.

    Science.gov (United States)

    von Trotha, Jakob W; Campos-Ortega, José A; Reugels, Alexander M

    2006-04-01

    Neurulation in zebrafish (Danio rerio) embryos is characterized by oriented cell divisions and the progressive establishment of cellular polarity. Mitoses in the neural plate and neural tube are planar, but in the neural keel/rod stage, the mitotic spindle rotates by 90 degrees, causing cell divisions to occur perpendicular to the plane of the neuroepithelium. The mechanisms and molecules that establish cellular polarity and cause the stereotypic orientation of the mitotic spindle during neurulation are largely unknown. In Caenorhabditis elegans and Drosophila, the PAR/aPKC complex has been shown to be involved in both establishment of cellular polarity and spindle orientation. Here, we show that the conserved N-terminal oligomerization domain (CR1) and the PDZ domains of ASIP/PAR-3:EGFP are involved in its localization to the apical membrane in zebrafish neuroepithelial cells. We further show that the C-terminal part of ASIP/PAR-3 contributes to proper localization and that the apical localization signals in ASIP/PAR-3 prevent the basolateral localization of a Numb:PAR-3 fusion protein. The parallel orientation of the mitotic spindle in the neural tube, however, is only weakly impaired upon overexpression of various ASIP/PAR-3:EGFP constructs.

  12. Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer.

    Science.gov (United States)

    Zeng, Fang; Li, Zicong; Cai, Gengyuan; Gao, Wenchao; Jiang, Gelong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2016-10-01

    Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.

  13. Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1.

    Directory of Open Access Journals (Sweden)

    Marta Mattiuzzo

    Full Text Available BACKGROUND: Highly Expressed in Cancer protein 1 (Hec1 is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells. METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells. CONCLUSIONS/SIGNIFICANCE: Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore

  14. 人IL-37b基因真核表达载体的构建与表达%Construction and expression of eukaryotic expression vector of human IL-37 b gene

    Institute of Scientific and Technical Information of China (English)

    姚静; 程江; 裴雪枫; 王静宇; 袁明

    2016-01-01

    目的:构建pEGFP-N1/IL-37b真核表达载体,并检测其在THP-1细胞中的表达情况。方法从人PBMCs中提取总RNA,利用RT-qPCR技术扩增出IL-37b基因编码区序列,克隆至pEGFP-N1真核表达载体,将构建的重组质粒pEGFP-N1/IL-37b转染到THP-1细胞中,通过RT-qPCR和Western blot检测IL-37的表达。结果双酶切及基因测序结果显示IL-37b基因正确插入真核表达载体pEGFP-N1中;RT-qPCR和Western blot结果显示转染THP-1细胞后,IL-37表达水平明显升高(P<0.01)。结论成功构建了新型抗炎因子IL-37真核表达载体pEG-FP-N1/IL-37b,为进一步研究IL-37功能及与相关疾病的关系奠定基础。%Objective To construct the eukaryotic expression vector pEGFP-N1/IL-37b and analyze the expression of IL-37 gene in THP-1 cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells ( PB-MCs) and the coding region of IL-37b gene was amplified by RT-qPCR. Then, the gene was cloned into pEGFP-N1 eu-karyotic expression vector. After transfected the recombinant plasmid into THP-1 cells, the expression of IL-37 was detec-ted by RT-qPCR and Western blot. Results Double restriction enzyme digestion and gene sequencing showed that IL-37b gene was correctly inserted into the eukaryotic expression vector pEGFP-N1. RT-qPCR and Western blot showed that the IL-37 expression level was increased significantly (P<0. 01) after transfection in THP-1 cells. Conclusions We successful-ly constructed a novel anti-inflammatory cytokine IL-37 eukaryotic expression vector pEGFP-N1/IL-37b, which lays a foun-dation for further study on IL-37 functions and its association with related diseases.

  15. Construction of human Gax gene eukaryotic expression vector and its expression in vascular smooth muscle cells%人Gax基因真核表达载体的构建及在血管平滑肌细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    郑辉; 薛松; 连锋; 汪永义

    2011-01-01

    AIM: To construct human Gax eukaryotic expression vector of pEGFP-N1-Gax, and observe its expression in the rabbit vascular smooth muscle calls (VSMCs). METHODS: human Gax cDNA was obtained by polymerase chain reaction(PCR) from pCMV-SPORT6-Gax plasmid. After digested with Nhe l and Xho l, the PCR product was cloned into eukaryotic expression vector pEGFP-N1 of green fluorescent protein(GFP) reported gene encoding green fluorescence protein, and then the recombinant plasmid pEGFP-N1-Gax was transfected into rabbit VSMCs using Sofast after it was identified by restriction enzyme digestion analysis and gene sequencing. Human Gax expression in rabbit VSMCs was detected by examining GFP expression of transfected cells under fluorescence microscope and RT-PCR. RESULTS: Agarose gel electrophoresis detection showed that human Gax DNA segment was about 915 bp, which accorded with the expectation. The restriction enzyme digestion analysis and DNA sequencing assays of recombinant vector pEGFP-N1-Gax showed the correct orientation and sequence. The expression of GFP in rabbit VSMCs transfected with pEGFP-N1-Gax was observed by fluorescence microscopy, and the expression of human Gax mRNA was confirmed by RT-PCR. CONCLUSION; The recombinant plasmid pEGFP-N1-Gax is successfully constructed, and expressed positively in rabbit VSMCs, it provide experimental basis to study the effects of Gax gene on cardiovascular disease.%目的:构建人Gax基因真核表达载体,并观察在兔血管平滑肌细胞中的表达.方法:通过PCR从pCMV-SPORT6-Gax质粒中扩增出人Gax cDNA片段,经双酶切后装入到有绿色荧光蛋白报告基因的真核表达载体pEGFP-N1中,经限制性内切酶酶切分析和DNA测序鉴定后通过梭华-Sofast转染试剂介导重组质粒转染至兔血管平滑肌细胞中进行表达.通过荧光显微镜观察转染细胞的绿色荧光蛋白表达和RT-PCR扩增转染细胞的cDNA来鉴定Gax在兔血管平滑肌细胞中的表达.结果:琼

  16. Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

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    Shaoxing YANG

    2012-12-01

    Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

  17. Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

    Institute of Scientific and Technical Information of China (English)

    JI Guang-hui; ZOU Jing-zhi; QU Le; YUE Ying; KUAI Jian-ke

    2004-01-01

    Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell lineTca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E. coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFPN3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N3, and then transferred into E. coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFPN3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60 % of the total amount. Conclusion: pFGFP- tk- IRES- IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be

  18. Development of a Canine Adenovirus Type 1 Vaccine Strain E3-deleted Based Expression Vector%犬腺病毒1型疫苗株E3缺失表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    黎皓; 唐七义; 张云; 王树蕙; 郭彩云

    2001-01-01

    Objective To evaluate canine adenovirus type 1 vaccine strain (Cannaught Laboratory Limited,CLL) as recombinant vaccine and gene transfer vector. Methods Recombinant virus CLLEGFP which contains enhanced green fluorescent protein(EGFP) reporter gene was constructed. CLLEGFP was used to infect various human derived cell lines (293, Hela, CO, SW, Hep-2 and CAM) by inoculating intraperitoneally(IP), intravenously(IV)and intramuscularly (IM)to Kunming mice other than oral administration. Various tissue samples of the mice were collected at multitime point for observing EGFP green fluorescence. Anti-EGFP antibodies were detected by Western blot analysis in the sera after 4 weeks. Results CLLEGFP can infect various human derived cell lines and express EGFP. EGFP green fluorescence were observed in liver tissue cells after IP transducing 3 days. All immune inoculation ways above could induce Kunming mice producing anti-EGFP antibodies which were identified by Western blot analysis. Conclusions These resluts indicate that CLL possess powerful potential as recombinant vaccine and gene transfer vector.%探索以犬腺病毒1型疫苗株(Cannaught Laboratory Limited.CLL)作为病毒重组疫苗和基因转移载体的可行性。方法构建带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的E3缺失重组病毒CLLEGFP。将CLLEGFP感染各种人源细胞,并以灌胃、腹腔注射、尾静脉注射和肌肉注射等不同途径接种昆明小鼠。多时间点取小鼠组织标本,冷冻干燥切片,观察EGFP的表达。4周后采集小鼠血清,以Western blot分析抗EGFP 抗体的产生。结果 CLLEGFP能够感染各种人源细胞并表达EGFP。在腹腔接种CLLEGFP 3 d的小鼠肝组织细胞中可见转导的EGFP。Western blot分析显示,以各种途径免疫接种重组病毒4周后的小鼠血清中均存在抗EGFP特异抗体。结论 CLL具有开发成为病毒重组疫苗和基因转移载体的潜力。

  19. Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

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    Kofuji Takefumi

    2011-05-01

    Full Text Available Abstract Background Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn. Results To develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC. Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP under the control of the preproenkephalin (PPE gene (penk1 promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side compared with the contralateral (non-ligated side. These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors. Conclusions These results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA, and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and

  20. Use of the PSA enhancer core element to modulate the expression of prostate- and non-prostate-specific basal promoters in a lentiviral vector context.

    Science.gov (United States)

    Chapel-Fernandes, S; Jordier, F; Lauro, F; Maitland, N; Chiaroni, J; de Micco, P; Mannoni, P; Bagnis, C

    2006-10-01

    Composite promoters combining the prostate-specific antigen (PSA) enhancer core element with promoter elements derived from gene coding for human prostate-specific transglutaminase gene, prostate-specific membrane antigen gene, prostate-specific antigen, rat probasin or phosphoglycerate kinase were characterized for their ability to specifically express the enhanced green fluorescent protein (EGFP) gene in prostate versus non-prostate cancer cell lines when transferred with a human immunodeficiency virus-1-based lentiviral vector. By themselves minimal proximal promoter elements were found to inefficiently promote relevant tissue-specific expression; in all the vectors tested, addition of the PSA enhancer core element markedly improved EGFP expression in LnCaP, a cancer prostate cell line used as a model for prostate cancer. The composite promoter was inactive in HuH7, a hepatocarcinoma cell line used as a model of neighboring non-prostate cancer cells. Among the promoters tested, the combination of the PSA enhancer and the rat probasin promoter showed both high specificity and a strong EGFP expression. Neither a high viral input nor the presence of the cPPT/CTS sequence affected composite promoter behavior. Our data suggest that composite prostate-specific promoters constructed by combining key elements from various promoters can improve and/or confer tissue specific expression in a lentiviral vector context.

  1. 脂肪组织特异性表达载体的构建%Construction of Adipose Tissue - specific Expression Vector

    Institute of Scientific and Technical Information of China (English)

    华晓敏; 许登高; 潘庆杰

    2012-01-01

    采用PCR技术克隆了小鼠脂肪组织特异表达的脂肪酸结合蛋白ap2基因增强子和启动子,通过DNA重组技术将该基因增强子和启动子重组于pEGFP - N1真核表达载体上,构建pEGFP - N1 - ap2重组质粒,通过PCR扩增、酶切电泳分析和测序的方法对重组质粒进行鉴定,并转染小鼠前脂肪细胞,通过荧光素酶活性检测特异性表达强度.结果表明,本实验克隆的ap2基因增强子和启动子的碱基组成与GenBank中的ap2基因序列完全一致,通过DNA重组技术将该基因增强子和启动子重组于pEGFP- N1真核表达载体上,成功构建了脂肪组织特异表达的重组质粒.为以后的转基因动物的研究奠定了基础.%The mouse adipose tissue -specific fatty acid binding protein ap2 gene enhancer /promoter was amplified by PCR amplification, and it was recombined into pEGFP - Nl eukaryotic expression vector by recombinant DNA technology, to obtain pEGFP - Nl - ap2 recombinant plasmid, which was identified by PCR amplification, enzyme digestion and DNA sequencing and infected with mouse pre - adipocytes, and its expression was detected by the fluorescence detection of the enzyme activity specific expression strength. The results showed that, cloned gene enhancer and promoter is consistent with the ap2 gene sequences in GenBank. The enhancer / promoter was recombined into pEGFP - Nl eukaryotic expression vector by recombinant DNA technology. The construction of the adipose tissue - specific expression vector was successfully constructed, which can provide a necessary basis for further study.

  2. Expression of non-structural protein NS3 gene of Bombyx mori densovirus (China isolate)

    Institute of Scientific and Technical Information of China (English)

    Huijuan Yin; Qin Yao; Zhongjian Guo; Fang Bao; Wei Yu; Jun Li; Keping Chen

    2008-01-01

    The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate),named BmDNV-3,is a kind of bidensovirus.It is a new type of virus with unique replication mechanisms.To investigate the effects of the NS3 gene during viral DNA replication,a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate).Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe,respectively.The NS3 protein was expressed in Escherichia coli BL21.The pFastBacHTe-NS3 was transformed to E.coli DH10Bac.The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3)isolated from the white colonies were transfected into BraN-4 cells using a transfection reagent.BmN-4 cells were infected with recom-binant virus to express fusion proteins.The expression of fusion protein around 30 kDa in E.coli BL21 was identified by SDS-PAGE,Western blotting,and mass spectrometry.The expressed NS3 protein by B.mor/nucleopolyhedrovirus bacmid system was confirmed byWestern blotting using an anti-NS3 polyclonal antibody.And about 45 kDa protein was found.The expressed fusion protein was smalleithan the expected size of EGFP-NS3,55 kDa.Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected lar-vae with smaller molecular size.

  3. Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

    Science.gov (United States)

    Koelzer, Stefan; Kölsch, Yvonne; Panfilio, Kristen A

    2014-01-01

    The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the

  4. Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

    Directory of Open Access Journals (Sweden)

    Stefan Koelzer

    Full Text Available The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid. Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control

  5. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

    Energy Technology Data Exchange (ETDEWEB)

    Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong; Orr, Galya; Xiong, Yijia; Squier, Thomas; Rorrer, Gregory L.; Roesijadi, Guritno

    2016-01-08

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively bound the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.

  6. Cloning of the Eukaryotic Expression Vector with Nerve Growth Factor in Rats and Its Effects on Proliferation and Differentiation of Mesencephal Neural Stem Cells of Fetal Rats

    Institute of Scientific and Technical Information of China (English)

    Minhua LIN; Lin YANG; Rong FU; Hongyang ZHAO

    2008-01-01

    Summary: The eukaryotic expression vector containing full-length cDNA sequence of rate nerve growth factor (NGF) β subunit was constructed and its effects on proliferation and differentiation of neural stem cells were observed. By using PCR, full-length cDNA sequence of NGF β subunit in rats was cloned and ligated into the eukaryotic expression vector pEGFP-N1-NGE The recombinant plasmid pEGFP-N1-NGF was transfected into the mesencephal neural stem cells of embryonic rats by Lipofectamin and transiently expressed. MTT method was used to determine the effects of NGF on proliferation of neural stem cells, and under phase-contrast microscopy, the effects of NGF on growth of nervous processes following differentiation of neural stem cells were observed. Sequence analysis indicated that the cloned full-length cDNA sequence of rat NGF β was identical to that of published sequence encoding NGF in gene GeneBank. The transfection of recombinant plasmid pEGFP-N1-NGF into mesencephal neural stem cells of embryonic rats could obviously promote proliferation of neural stem cells and faciliate the growth of neural stem cells-derived nerve cells. It was suggested that neural stem cells could be used as a vehicle of gene transfer, and the expression of NGF β subunit in the neural stem cells could promote the growth of nerve cells derived from neural stem cells.

  7. Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection.

    Science.gov (United States)

    Jheng, Yu-Hsuan; Lee, Lin-Han; Ting, Chen-Hung; Pan, Chieh-Yu; Hui, Cho-Fat; Chen, Jyh-Yih

    2015-01-01

    Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 μg/100 μl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

  8. Ang2基因RNA干扰慢病毒表达载体的构建与鉴定%Construction and Identification of RNAi Lentiviral Expression Vector for Ang2 Gene

    Institute of Scientific and Technical Information of China (English)

    王彪; 张炜强; 单秀英; 刘照亮; 郭国祥; 庄福连

    2011-01-01

    Objective To construct and identify the RNAi lentiviral expression vector for angiopoietin ( Ang )2 gene, and identify its validity. Methods pSilenser 1.0-U6 - Ang2-siRNA reccombinant plasmid to ploymorphonuclear neutrophilic leukocyte-effective green fluorescence protein( pNL-EGFP )vector, which were digested and electorphoresis identified by using enzyme Xha Ⅰ , were joined to get pNL-EGFP-U6-Ang2 -siRNA lentiviral transfer plamid , then the PNL-EGFP-U6-Ang2-siRNA lentiviral transfer plasmid , vesicular stomatitis virus G-protein envelope plasmid and pHelper packaging plasmid were cotransfected into 293T cells , resulting in lentivirus. The virus supernatant were collected and the viral titer was determined.Results The lentiviral transfer plasmids of pNL-EGFP-U6-Ang2 -siRNA were constructed successfully and identified by using enzyme digestion electrophoresis of Xba Ⅰ and sequencing analysis,proved the correctness of the inserted Ang2-siRNA nudeotide sequence. EGFP-Ang2-siRNA virus were produced hy using the three plasmid lentiviral packaging system. The virus supernatant was collected and viral titers measured for the 9 ×103/μL. Conclusion The RNAi lentiviral expression vectors for Ang2 gene were constructed successfully ,and would be useful for the further research of the next research of interfering the Ang2 expression in malignant melanoma in vivo and vitro.%目的 构建促血管生成素(Ang)2基因的RNAi慢病毒表达载体,并鉴定其正确性.方法 将经XbaⅠ酶切电泳鉴定的pSilencer 1.0-U6-Ang2-siRNA重组质粒与经XbaⅠ酶切电泳鉴定的嗜中性多形核白细胞-绿色荧光蛋白转移质粒(pNL-EGFP)载体连接,产生pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒,再以pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒、水疱性口炎病毒G蛋白包膜质粒和包装质粒三质粒共转染293T细胞产生慢病毒,收集病毒上清液并测定病毒滴度.结果 成功构建pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒2条,通

  9. Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice

    Directory of Open Access Journals (Sweden)

    Palm Kaia

    2009-06-01

    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF is a small secreted protein that has important roles in the developing and adult nervous system. Altered expression or changes in the regulation of the BDNF gene have been implicated in a variety of human nervous system disorders. Although regulation of the rodent BDNF gene has been extensively investigated, in vivo studies regarding the human BDNF gene are largely limited to postmortem analysis. Bacterial artificial chromosome (BAC transgenic mice harboring the human BDNF gene and its regulatory flanking sequences constitute a useful tool for studying human BDNF gene regulation and for identification of therapeutic compounds modulating BDNF expression. Results In this study we have generated and analyzed BAC transgenic mice carrying 168 kb of the human BDNF locus modified such that BDNF coding sequence was replaced with the sequence of a fusion protein consisting of N-terminal BDNF and the enhanced green fluorescent protein (EGFP. The human BDNF-BAC construct containing all BDNF 5' exons preceded by different promoters recapitulated the expression of endogenous BDNF mRNA in the brain and several non-neural tissues of transgenic mice. All different 5' exon-specific BDNF-EGFP alternative transcripts were expressed from the transgenic human BDNF-BAC construct, resembling the expression of endogenous BDNF. Furthermore, BDNF-EGFP mRNA was induced upon treatment with kainic acid in a promotor-specific manner, similarly to that of the endogenous mouse BDNF mRNA. Conclusion Genomic region covering 67 kb of human BDNF gene, 84 kb of upstream and 17 kb of downstream sequences is sufficient to drive tissue-specific and kainic acid-induced expression of the reporter gene in transgenic mice. The pattern of expression of the transgene is highly similar to BDNF gene expression in mouse and human. This is the first study to show that human BDNF gene is regulated by neural activity.

  10. Expression of matrix metalloproteinases-3 gene in lens epithelial cell and its significance%基质金属蛋白酶3在晶状体上皮细胞中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    杨晶晶; 何湘珍; 向浩天; 周晓敏; 王云; 蔡素萍

    2012-01-01

    Background Posterior capsular opacification(PCO) is common complication after extrecapsular extract of cataract.Matrix metalloproteinases-3 (MMP-3) can degrade all the extracellular matrix except polyose.The gene therapy of PCO upon MMP-3 is the researching hot topic.Fibronectin ( FN ) is a degrade gelatin,so its expression can reflect the effect of MMP-3 on LECs indirectly. Objective The aim of this study was to construct MMP-3 eukaryotic recombination plasmid and transfect to lens epithelium cells(LECs) for the observation of MMP3 expression,and to explore the feasibility of gene therapy for after cataract. Methods Six fresh lenses were obtained from pigs.LECs were cultured using explant method.The eukaryotic expression vector pEGFP-N1-MMP-3 was reconstructed with MMP-3 and pEGFP-N1 plasmids.The accuracy of MMP-3 gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-MMP-3 into LECs of pig,the expression of MMP-3 protein in the cells was indirectly observed by green fluorescent protein.The expression of FN in LECs was detected using Western blot. Results The result of double enzyme digestion was consistent with the base number of pEGFP-N1 plasmids and target fragment.By enlacing the result of DNA sequencing analysis with software,the resemblance of the DNA sequence of MMP-3 from recombination plasmid pEGFP-N1-MMP-3 and that of homo MMP-3 was 99.6%,indicating that the target fragment was inserted to pEGFP-N1 plasmids successfully.Green fluorescence for GFP was seen in the LECs in pEGFP-N1-MMP-3 transfected group,but absent response for GFP was in empty vector group.Western blot revealed that the relative expression level of FN in LECs was 0.666±0.008 in pEGFP-N1-MMP-3 trasfected group and 0.326 ±0.071 in empty vector group,with a significant difference between these two groups(P=0.000). Conclusions Eukaryotic recombination plasmid pEGFP-N1-MMP-3 is successfully constructed,and MMP-3 can be expressed in

  11. Fluorescent reporter signals, EGFP and DsRed, encoded in HIV-1 facilitate the detection of productively infected cells and cell-associated viral replication levels

    Directory of Open Access Journals (Sweden)

    Kazutaka eTerahara

    2012-01-01

    Full Text Available Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1 strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively-infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

  12. Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shengtao; LIU Wenli; HE Peigen; GONG Feili; YANG Dongliang

    2006-01-01

    In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

  13. Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

    Science.gov (United States)

    Katoh, A; Shoguchi, K; Matsuoka, H; Yoshimura, M; Ohkubo, J-I; Matsuura, T; Maruyama, T; Ishikura, T; Aritomi, T; Fujihara, H; Hashimoto, H; Suzuki, H; Murphy, D; Ueta, Y

    2014-05-01

    The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

  14. Imaging of Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression with Radiolabeled 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in Liver by Hydrodynamic-based Procedure

    Energy Technology Data Exchange (ETDEWEB)

    Song, In Ho; Lee, Tae Sup; Kang, Joo Hyun; Lee, Yong Jin; Kim, Kwang Il; An, Gwang Il; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-10-15

    Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynamic injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU.

  15. 稳定表达HLA-A*1101蛋白的K562细胞株的建立%Establishment of Stable Subline of K562 Cells Expressing Human Leucocyte Antigen A1101

    Institute of Scientific and Technical Information of China (English)

    查显丰; 周羽竝; 杨力建; 陈少华; 李萡; 闫小娟; 李扬秋

    2011-01-01

    The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A * 1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A* 1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3' terminal of the HLA-A' 1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A* 1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A' 1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A' 1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A' 1101-T2A-EGFP plasmid was successfully constructed; after G4I8 selection for 2 months, two sublines of K562 cells (HLA-A'1101 *K562, pEGFP-N3 * K562) expressing GFP were constructed. The expression of HLA-A * AJ101 gene could be determined in HLA-A* 1101 * K562 cell line by RT-PCR,while the pEGFP-N3 * K562 cells could not express HLA-A * A1101 gene. HLA-A* 1101 protein and GFP double positive HLA-A * 1101* K562 cells were upto 88. 5% , which was obviously higher than pEGFP-N3* K562 cells (0. 698% ) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A * 1101 * K562 cells has been established and a subline of K562 cell expressing HLA-A* 1101 protein on its cell membrane was successfully

  16. Construction and detection of expression vectors of microRNA-9a in BmN cells

    Institute of Scientific and Technical Information of China (English)

    Yong HUANG; Quan ZOU; Sheng-peng WANG; Shun-ming TANG; Guo-zheng ZHANG; Xing-jia SHEN

    2011-01-01

    MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21-23 nucleotides in length,which regulate gene expression by base-pairing with 3' untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a)precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.=miRNA-9a (miR-9a),EGFP gene,Bombyx mori N (BmN) Cells,Expression vector

  17. The improvement of liposome-mediated transfection of pEGFP DNA into human prostate cancer cells by combining low-frequency and low-energy ultrasound with microbubbles.

    Science.gov (United States)

    Bai, Wen-Kun; Wu, Zuo-Hui; Shen, E; Zhang, Ji-Zhen; Hu, Bing

    2012-02-01

    The aim of this study was to explore the use of a contrast agent to study the effects of exposure to ultrasound, in combination with microbubbles, on liposome-mediated transfection of genes into human prostate cancer cells. A contrast agent was used to study the effects of ultrasound exposure in combination with microbubbles on liposomes, which transfect genes into human prostate cancer cells. The human prostate cancer cell line PC-3 in suspension was exposed to ultrasound with a 20% duty cycle (i.e., 2 sec 'on' time and 8 sec 'off' time) lasting 5 min, with and without ultrasound contrast agent (SonoVue™) using a digital sonifier at a frequency of 21 kHz and an intensity of 4.6 mW/cm2. Immediately after exposure to ultrasound, cell viability and membrane damage were measured. After exposure to ultrasound, the cell suspensions were put into 12‑well plates and cultured for 24 h. Fluorescence microscopy and flow cytometry were used to detect pEGFP transfection efficiency. Exposure to ultrasound alone and ultrasound combined with microbubbles resulted in minimal cell death and induced negligible cell membrane damage. Ultrasound combined with microbubbles had a greater effect on cell membrane damage in all groups: the average cell membrane damage was 41.87%, and it was approximately 42‑fold greater than in the control group. The average transfection efficiency of PC-3 cells was 20.30% for the liposome (Lipofectamine™)+pEGFP+ultrasound+ultrasound contrast agent (SonoVue) group; this was the highest rate of all groups measured and was approximately 81‑fold greater than that of the control group. The use of low-frequency and low-energy ultrasound, in combination with microbubbles, could be a potent physical method for increasing liposome gene delivery efficiency. This technique is a promising non-viral approach that can be used in prostate cancer gene therapy.

  18. Cloning, Expression and Characterization of Zebra Fish Ferroportin in Hek 293T Cell Line

    Directory of Open Access Journals (Sweden)

    A Memarnejadian

    2012-01-01

    Full Text Available Background: Ferroportin (Fpn, a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms.Methods: To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP-N1. The final resulted plasmid (pEGFP-ZFpn was used for expression of Fpn-EGFP protein in Hek 293T cells.Results: The expression was confirmed by appearance of fluorescence in Hek 293 T cells. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model, NetOGlyc 3.1 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids.Conclusion: The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies.

  19. Production of pigs expressing a transgene under the control of a tetracycline-inducible system.

    Directory of Open Access Journals (Sweden)

    Yong-Xun Jin

    Full Text Available Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP. At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.

  20. The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

    Science.gov (United States)

    Kim, Jong-Hyun; Lee, Sang-Hee; Sohn, Hae-Jin; Lee, Jinyoung; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

    2012-12-01

    The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

  1. PRP基因绿色荧光蛋白载体构建及其生物学功能初步研究%Construction of the eukaryotic expression vector of proliferin related protein and preliminary study on its biological activity in 293FT cells

    Institute of Scientific and Technical Information of China (English)

    王琦; 郝杰; 赵丽娜; 吕自兰; 王丽敏; 余秋波; 王应雄; 黎刚

    2011-01-01

    目的:构建Prolifein related protein(PRP)基因荧光表达载体,并初步探讨其生物学功能.方法:提取小鼠睾丸RNA,RT-PCR扩增PRP基因编码区片段,酶切连入pEGFP-C1载体,构建含有PRP基因片段的绿色荧光表达载体PRP-pEGFP-C1.为了探讨PRP基因功能,PRP-pEGFP-C1经Lipofectamine2000介导转染293FT细胞,MTT实验分析细胞增殖变化,流式细胞仪分析细胞周期分布.结果:质粒PRP-pEGFP-C1经测序验证,证实PRP基因已正确连入pEGFP-C1载体.免疫荧光染色显示PRP 定位于293FT细胞胞浆.MTT结果表明,上调PRP基因引起293FT细胞增殖活性下降,细胞周期分布情况提示G1期细胞增加,S期细胞减少.结论:成功构建PRP-pEGFP-C1荧光表达载体,对其功能研究表明PRP具有抗人293FT细胞增殖,扰乱细胞增殖周期的功能.本研究为PRP在抗人肿瘤方面的研究提供基础.%Objective : To construc:t the eukaryotie expression vector of proliferin related protein , and to investigate its biological function. Methods : The coding region of proliferin related protein amplified hy RT-PCR from mouse testis RNA was cloned into the pEGFP-C1 vector to form the recomhinant PRP-pEGFP-C1 plasmid. Then the recomhinant plasmid was transfected int0 293FT cells , and the expression of PRP was detected by RT-PCR and Western blotting. MTT and flow cytometry ( FCM ) analysis were employed to analysis the cell proliferation activity and the cell cycle , respectively. Results : PRP coding region was successfully cloned into the eukaryotic vector pEGFP-C1. The recomhinant vector was transfected into 293FT , results of RT-PCR and Western blotting showed that the PRP could express in 293FT cells. Overexpressing PRP gene decreased the cell proliferation activity as well as disturbing the cell cycle distrihution of 293FT cells in vitro. Conclusion : We have constructed a recombinant vector expressing PRP and eGFP fusion protein , which is in favour of the further study on the gene

  2. 电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响%Effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogene-sis of distraction area during early mandibular distraction osteogenesis

    Institute of Scientific and Technical Information of China (English)

    吴国平; 黎德平; 何小川; 李盛华; 杨智慧; 廖毅; 郭力

    2010-01-01

    目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组:质粒+电穿孔组(A组),质粒组(B组),生理盐水+电穿孔组(C组),空白对照组(D组).各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.%Objective To explore the effect of electroporation mediated gene therapy on angiogene-sis of the distraction area during early mandibular distraction osteogenesis (DO). Methods Thirty-two New-Zeland rabbits were randomly divided into 4 groups: group A: recombinant plasmid pIRES-VEGF165-EGFP and electroporation; group B: recombinant plasmid pIRES-VEGF165-EGFP; group C: normal saline (NS) and electroporation and group D: control group. The rabbits were sacrificed at 1d, 3d, 7d and 14d after injection, respectively. The distraction area tissue was removed for histological examination and electron microscopy, and immunohistochemical stain for CD34 was performed to detect the microvessel density. Results Generation of vascular endothelial cells (VEC) in the group A and group B were active, and majority of VEC in groups C and D took on early change of cataplasia and apoptosis. The

  3. Estblishment of Porcine Oct-4-EGFP Site-directed Transgene Cell Lines using Engineered TALENs%利用TALENs技术建立基因定点修饰的猪Oct-4-EGFP细胞系

    Institute of Scientific and Technical Information of China (English)

    刘畅; 冯冲; 宋志强; 李西睿; 王宁; 储明星; 潘登科

    2013-01-01

    本研究旨在建立一个用于干细胞示踪的报告体系,将EGFP cDNA及两侧带有LoxP位点的neo抗性基因插入五指山小型猪内源性Oct-4基因的终止密码子处,以Oct-4基因完整的5'调控区启动EGFP的表达,为猪干细胞研究提供有价值的工具.试验定制了针对猪Oct-4终止密码子的TALENs,与打靶载体共转染猪耳成纤维细胞,药物筛选得到抗性克隆点514个,经过PCR鉴定,共获得杂合阳性克隆点36个,打靶效率分别为5.6%和13.0%.本研究成功获得了Oct-4-EGFP转基因细胞系,并证明了TALENs技术可以明显提高同源重组效率.

  4. Propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.

    Science.gov (United States)

    Jónsson, Stefán R; Andrésdóttir, Valgerdur

    2011-10-09

    Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis. The primary target cells of MVV in vivo are considered to be of the monocyte lineage. Certain strains of MVV can replicate in other cell types, however. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have nserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages. There is good correlation between measurements of TCID₅₀ and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo.

  5. Antitumor effect of COOH-terminal polypeptide of human TERT is associated with the declined expression of hTERT and NF-κB p65 in HeLa cells.

    Science.gov (United States)

    Wu, Xian; Chen, Jiasheng; Cao, Ying; Xie, Baoping; Li, Hongwei; Zhou, Pingzheng; Qiu, Yuchang; Pang, Jianxin

    2015-12-01

    Human telomerase reverse transcriptase (hTERT) plays an important role in the development of tumors and has been investigated as a potent target for anticancer therapy. In the present study, we constructed a recombinant adenovirus, Ad-EGFP-C197 which was capable of expressing COOH‑terminal polypeptide of hTERT (amino acid 936-1,132, termed as C197 for the reason that it contains 197 amino acids). Infection of HeLa cells with Ad-EGFP-C197 suppressed the activity of telomerase, decreased the expression of hTERT and NF-κB p65, and induced rapid growth delay and apoptosis of HeLa cells in vitro. In nude mice xenografted with HeLa tumors, injection of Ad-EGFP-C197 into the tumor nodule significantly slowed tumor growth and promoted tumor cell apoptosis, as well as reduced the expression of NF-κB p65 in tumor tissues. In the present study, we suggest that the antitumor effect of C197 is associated with the declined expression of hTERT and NF-κB p65. Our results highlight the potential of C197 in tumor therapy.

  6. Expression liver-directed genes by employing synthetic transcriptional control units

    Institute of Scientific and Technical Information of China (English)

    Marie-Luise Lemken; Wolfgang A. Wybranietz; Ulrike Schmidt; Florian Graepler; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer

    2005-01-01

    AIM: To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver,thereby compensating for the lack of specificity of currently available gene therapeutic vector systems.METHODS: Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS: A new bifunctionally-enhanced green fluorescent protein (EGFP)/neor fusion gene cassette was generated,and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis.CONCLUSION: Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver

  7. 肝片吸虫GST真核表达载体构建及重组蛋白活性分析%Construction on eukaryotic expression plasmid of Fasciola hepatica GST gene and analysis on recombinant protein

    Institute of Scientific and Technical Information of China (English)

    冉旭华; 闻晓波; 王春仁; 刘娣; 孙中武; 李晓娟; 王密

    2011-01-01

    目的 构建肝片吸虫谷胱甘肽S-转移酶(GST)的真核表达载体,研究重组蛋白的免疫原性.方法 以构建好的重组质粒pET30a-FhGST为模板,利用PCR技术扩增肝片吸虫谷胱甘肽S-转移酶基因(GST),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-GST,转染Hela细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况.结果 重组质粒pEGFP-GST在Hela细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应.结论 肝片吸虫GST真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有生物学活性,可做为分子疫苗的候选进行进一步的研究.%In this research, we constructed eukaryotic expression plasmid of Fasciola hepatica GST gene and analyzed the immunogenicity of recombinant protein. The glutathione S-transferase (GST) gene of F. hepatica played the importanty protective role against the worms infection. In this research,GST was amplified by PCR from the pET30a-FhGST, and then inserted into pEGFP-Nl vector to construct recombinant plasmids pEGFP-GST. The recombinant plasmid pEGFP-GST was transfected into Hela cells and fluorescent signal was detected by fluorescence microscope. Western blotting analysis was done to analysze immunogenicity of recombinant protein. And the results demonstrated that eukaryotic expression plasmid of Fasciola hepatica GST gene was constructed successfully. Recombinant protein could be specifically recognized by goat serum infected by Fasciola hepatica, which proving its immunoreactivity. It's suggested that the eukaryotic expression plasmid might be used as gene vaccine in further research.

  8. Heterologous Expression of Rat Testis GABAA Receptor β3t Splicing Variant in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    Shi-feng LI; Yu-guang CHEN; Yuan-chang YAN; Yi-ping LI

    2004-01-01

    Objective To characterize a possible retention function of unique sequence in the 5'end of rat testis GABAA receptor β3t splicing variantMethods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined.Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively. The chimera product failed to be translocated into the cell surface when expressed in CHO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane.Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.

  9. Expression of the nfa1 gene cloned from pathogenic Naegleria fowleri in nonpathogenic N. gruberi enhances cytotoxicity against CHO target cells in vitro.

    Science.gov (United States)

    Jeong, Seok-Ryoul; Lee, Sang-Chul; Song, Kyoung-Ju; Park, Sun; Kim, Kyongmin; Kwon, Myung-Hee; Im, Kyung-Il; Shin, Ho-Joon

    2005-07-01

    The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5' upstream regions, the nfa1 open reading frame, and 3' downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

  10. Construction and identification of recombination expression vector Ksp-Cadherin-Gpx1-Klk1

    Institute of Scientific and Technical Information of China (English)

    解立怡; 薛武军; 项和立; 麻孙凯

    2008-01-01

    Objective To construct and identify the Gpx1-Klk1 vector which contains kidney-specific promoter (Ksp-cadherin). Methods Through PCR amplification, the human Gpx1, Klk1, and Ksp-cadherin cDNA were obtained by taking Gpx1 cDNA, Klk1 cDNA, and Ksp-cadherin BAC as templates. After being testified, the PCR products were inserted into the expressive vector pIRES-EGFP step-by-step to produce a recombinant vector Ksp-cadherin-Gpx1-Klk1. This vector was examined by restriction enzyme digestion and sequence analysis...

  11. hLMO3基因真核表达载体的构建及蛋白的表达和定位%Construction of eucaryotic plasmid of human LMO3 Gene and the expression and localization of fusion protein

    Institute of Scientific and Technical Information of China (English)

    顾卉; 佟宇鑫; 刘彤; 李妍; 李丹妮; 袁正伟

    2011-01-01

    Objective To construct the expression plasmid of human LIM domain only 3 (hLMO3) gene and identify the expression and localization of its fusion protein in human HEK293 cells. Methods The hLMO3 coding sequence was amplified by polymerase chain reaction by using cDNA library derived from human fetal brain as the template and subcloned into pEGFP vector. After the target region was identified by enzyme digestion and sequencing, the plasmid was transfected into HEK293 cells. The expression of the recombinant plasmid in HEK293 cells was detected by Western blot. The localization of pEGFP-LMO3 in HEK293 cells was observed with laser scanning confocal microscopy. Results hLMO3 was constructed into the expressing vector pEGFP successfully, the length of the fragment identified by restriction enzyme digestion was 440bp. The expression of pEGFP-LMO3 fusion protein with a molecular weight of 42 kDa was detected by Western blot in human HEK293 cells. The pEGFP-LMO3 fusion protein was mostly localized in the nucleus of HEK293 cells. Conclusion The recombinant plasmid of hLMO3 gene was successfully cloned into eukaryotic expressing vector, and the pEGFP-LMO3 fusion protein was mostly localized in the nucleus of HEK293 cells.%目的 构建hLMO3真核表达载体并证实融合蛋白在细胞内的表达及定位.方法 以人胎脑文库cDNA为模板,PCR扩增hLMO3基因cDNA全长,亚克隆至pEGFP表达载体中.将构建的重组质粒进行酶切测序鉴定,并转染到人上皮细胞HEK293细胞中,提取细胞蛋白进行Western blot检测.利用激光扫描共聚焦显微镜观察pEGFP-hLMO3在HEK293细胞内的定位.结果 hLMO3基因cDNA全长克隆到了真核表达载体pEGFP中,酶切鉴定片段为440 bp;Western blot检测到融合蛋白在HEK293细胞表达,分子量约为42kDa,pEGFP-LMO3在人HEK293细胞中主要定位于细胞核内.结论 成功构建了hLMO3基因cDNA全长的真核表达载体,pEGFP-LMO3蛋白在HEK293细胞中主要定位于细胞核内.

  12. Experimental study on efficacy of bone marrow mesenchymal stem cell modified by plasmid plRESneo-EGFP-BDNF in treatment of rats with Parkinson's disease%移植脑源性神经生长因子修饰骨髓间充质干细胞对帕金森病大鼠模型治疗作用的实验研究

    Institute of Scientific and Technical Information of China (English)

    赵钢勇; 张平; 赵慧新; 代瑞廷; 张本恕

    2013-01-01

    Objective:To investigate efficacy of bone marrow mesenchymal stem cells(MSCs) modified by plasmid pIRESneo-EGFP-BDNF in treatment of rats with Parkinson's disease(PD). Methods:pIRESneo-EGFP-BDNF plasmid was established by reconstructing pEGFP(N1)-BDNF plasmid and bone marrow MSCs was transfected by electroproation. PD rat models were set up by 6-OHDA and then divided into four groups randonly:sham group,PD group,MSCs group and brain-derived neurotrophic factor(BDNF) group. Rotating behaviors of rats were induced by intraperitoneal injection of Apomorphine at 2,4,8 weeks after bone marrow MSCs transplantation or MSCs modification of plasmid pIRESneo-EGFP-BDNF through cerebral lateral ventricle. BDNF protein and tyrosine hydroxylase(TH) protein in mesencephalic nigra were measured by Western blot. Results:Plasmids pIRESneo-EGFP-BDNF were constructed successfully,which was identified by double digestion. Rotation numbers were significantly decreased in MSCs group and BDNF group than in PD group at 2,4,8 weeks after the transplantation(P<0.05),with more evident improvement in BDNF group than in MSCs group(P<0.05). Levels of BDNF protein and TH protein in nigra at 8 weeks after PD model being intervened by cell transplantation were increased significantly in BDNF group than in MSCs group and PD group. Conclusions:Transplantation of pIRESneo-EGFP-BDNF modified MSCs through cerebral lateral ventricle can improve PD rats' behavior and increase BDNF and TH protein levels.%目的:观察移植pIRESneo-EGFP-BDNF修饰骨髓间充质干细胞(mesenchymal stem cells,MSCs)对帕金森病(Parkinson's disease,PD)大鼠的研究.方法:将pEGFP (N1)-BDNF质粒进行改造与pIRESneo相连结,构建高拷贝质粒pIRESneo-EGFP-BDNF,采用电穿孔法转染骨髓MSCs;制备PD大鼠模型,随机分为假手术组、PD组、MSCs组、脑源性神经生长因子(brain-derived neurotrophic factor,BDNF)组,经侧脑室移植MSCs或pIRESneo-EGFP-BDNF修饰骨髓MSCs术后2、4、8周,腹

  13. 转增强型绿色荧光蛋白的脐血干细胞制备嵌合体小鼠的研究%The chimeric mice derived from umbilical cord blood stem cells of EGFP-transgenic mouse

    Institute of Scientific and Technical Information of China (English)

    段彪; 杜海燕; 张荣

    2013-01-01

    Objective The chimeric mice were prepared by microinjection of blastocyst cavity using umbilical cord blood stem cells(UCBSCs) of Enhanced Green Fluorescent Protein(EGFP)-transgenic mouse, which was expected to provide a theoretical and experimental basis for the study of in-vivo differentiation of adult stem cells. Methods Mouse UCBSCs expressing green fluorescence was microinjected into blastocyst cavity and several blastocysts were transferred into uterus of pseudo pregnant mouse. First of all, new-born candidate chimeric mouse were observed through feather color. Secondly, the genomic DNA and total RNA were extracted to analyze chimeric rate in several tissues. Finally, flow cytometry was used to detect percentage of green fluorescent cells mice in several tissues. Results The UCBSCs expressing green fluorescent protein were successfully isolated. After flow cytometry analysis, the proportion of cells expressing green fluorescence was 80.25%. Through microinjection and embryo transfer, we got five white new-born mice and no chimeric feather color was observed. The analyses of PCR and RT-PCR were carried out to detect EGFP gene using six tissues including, heart muscle, liver, lung, skin, leg muscle and adipose tissue. The results showed that the leg muscle and adipose tissue of two mice were positive and the other tissues and six tissues of the other 3 mice were all negative. The leg muscle and adipose tissue of two positive mice were digested into single-cells suspension and were carried out flow cytometry analysis. The Results showed that the average chimeric rates of leg muscle and adipose tissue of two positive mice were 9.87% and 5.78%, respectively. Conclusion The results demonstrated that adult UCBSCs could differentiate into leg muscle and adipose tissue in vivo.%目的:本研究通过囊胚腔显微注射转增强型绿色荧光蛋白(EGFP)基因的脐血干细胞的方法制备嵌合体小鼠,以期为研究成体干细胞的体内分化提

  14. Construction and expression of attenuated salmonella typhimurium carrying human adiponectin gene eukaryotic expression vector%重组人脂联素基因在减毒沙门氏菌中的表达

    Institute of Scientific and Technical Information of China (English)

    周静; 周洲; 吴莹; 向廷秀; 姜政; 王丕龙

    2009-01-01

    目的:构建携带人脂联素(AdipoQ)基因真核表达载体并在减毒沙门氏菌中表达,为AdipoQ对非酒精性脂肪性肝病(NAFLD)的基因治疗提供依据.方法:从人脂肪组织中提取总RNA,通过实时荧光定量PCR(rRT-PCR)方法获得Adi-poQ基因并将其克隆到真核表达载体pEGFP-NI上,构建pEG-FP-N1-AdipoQ重组载体.重组质粒经鉴定后再电转入减毒沙门氏菌SL7207中表达.结果:克隆的人AdipoQ基因744 bp测序结果显示:1个碱基发生突变,654位:A→T,突变率为0.1%,氨基酸Glu→Asp.经SDS-PAGE,Western Blot检测融合蛋白Mr约为55×10~3(绿色荧光蛋白Mr约为27×10~3),能够被AdipoQ抗体识别.结论:成功构建携带AdipoQ基因真核表达载体的减毒沙门氏菌株,AdipoQ基因能够在减毒沙门氏菌中表达并与绿色荧光蛋白融合.为进一步研究其在NAFLD中的作用机制奠定了基础.%AIM: To construct and express an attenuated Salmonella typhimurium strain contraining human adiponectin gene eukaryotic expression vector, in order to lay the foundation for the genetic therapy of nonalcoholic fatty liver disease in future. METHODS: Total mRNA was extracted from human fat tissue and the adiponectin cDNA was obtained by RT-PCR, and then cloned into eukaryotic expression vector pEGFP-N1. The recombinant vector was identified, and transformed into attenuated Salmonella typhimurium strain SL7207, and the recombinant strain SL7207/pEGFP-NI-AdipoQ was screened by green fluorescent microscope and Western Blot. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes was found to be 744 bp, and had been inserted into eukaryotic expression vector, but as compared with gene reported by GenBank, 0. 1% of the gene mutation and O. 4% of amino acid residues change, respectively: 654 bp: A→T, amino acids: Glu→Asp. SDS-PAGE demonstrated that pEGFP-N1-AdipoQ was expressed in SL7207 strain, and the relatived molecular mass of the GFP-AdipoQ fusion protein

  15. SOX2 expression is upregulated in adult spinal cord after contusion injury in both oligodendrocyte lineage and ependymal cells.

    Science.gov (United States)

    Lee, Hyun Joon; Wu, Junfang; Chung, Jumi; Wrathall, Jean R

    2013-02-01

    The upregulation of genes normally associated with development may occur in the adult after spinal cord injury (SCI). To test this, we performed real-time RT-PCR array analysis of mouse spinal cord mRNAs comparing embryonic day (E)14.5 spinal cord with intact adult and adult cord 1 week after a clinically relevant standardized contusion SCI. We found significantly increased expression of a large number of neural development- and stem cell-associated genes after SCI. These included Sox2 (sex determining region Y-box 2), a transcription factor that regulates self-renewal and potency of embryonic neural stem cells and is one of only a few key factors needed to induce pluripotency. In adult spinal cord of Sox2-EGFP mice, Sox2-EGFP was found mainly in the ependymal cells of the central canal. After SCI, both mRNA and protein levels of Sox2 were significantly increased at and near the injury site. By 1 day, Sox2 was upregulated in NG2(+) oligodendrocyte progenitor cells (OPC) in the spared white matter. By 3 days, Sox2-EGFP ependymal cells had increased proliferation and begun to form multiple layers and clusters of cells in the central lesion zone of the cord. Expression of Sox2 by NG2(+) cells had declined by 1 week, but increased numbers of other Sox2-expressing cells persisted for at least 4 weeks after SCI in both mouse and rat models. Thus, SCI upregulates many genes associated with development and neural stem cells, including the key transcription factor Sox2, which is expressed in a pool of cells that persists for weeks after SCI.

  16. Expression of H5N1 influenza virus hemagglutinin protein fused with protein transduction domain in an alphavirus replicon system.

    Science.gov (United States)

    Yang, Shi-gui; Wo, Jian-er; Li, Min-wei; Mi, Fen-fang; Yu, Cheng-bo; Lv, Guo-liang; Cao, Hong-Cui; Lu, Hai-feng; Wang, Bao-hong; Zhu, Hanping; Li, Lan-Juan

    2010-01-01

    Alphavirus replicons, in which structural protein genes are replaced by heterologous genes, express high levels of the heterologous proteins. On the basis of the potencies of replicons to self-replicate and express foreign proteins and the remarkable intercellular transport property of VP22, a novel alphavirus Semliki Forest virus (SFV) replicon system of VP22 fused with a model antigen, hemagglutinin (HA), of the human-avian H5N1 influenza virus, was explored in this study. Further, replicon particles expressing HA, VP22, and enhanced green fluorescent protein (EGFP) individually were used as controls. By flow cytometry based on the analysis of transfection efficiency, SFV-EGFP replicon particle titer was 1.13 x 10(7)transducing units (TU)/ml. The titers of SFV-HA, SFV-VP22 and SFV-VP22-HA replicon particles, which were titrated by using SFV-EGFP replicon particles, were 1.42 x 10(7), 3.23 x 10(7), and 1.01 x 10(7)TU/ml, respectively. HA and VP22-HA expression was observed in SFV-HA- and SFV-VP22-HA-transfected BHK-21 cells, respectively. Immunofluorescence staining revealed that the fluorescence intensity in the SFV-VP22-HA-transfected BHK-21 cells was more than that in the SFV-HA-transfected BHK-21 cells. Both SFV-VP22-HA and SFV-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza. VP22-HA fusion protein with similar trafficking properties may also enhance vaccine potency.

  17. 喉癌来源的MAGE-A3基因真核表达载体及其稳定表达模型的构建和鉴定%Construction and identification of laryngocarcinoma-derived melanoma antigen-encoding-A3 gene eukaryotic expression vector and steady expression model

    Institute of Scientific and Technical Information of China (English)

    吉晓滨; 吕景礼; 陈敬贤; 刘启才; 谢景华

    2012-01-01

    目的:克隆人黑色素瘤相关抗原MAGE-A3基因,构建真核表达载体,检测、鉴定其在细胞中的表达.方法:MAGE-A3基因进行PCR扩增,凝胶回收PCR产物片段并连接至pMD18-T载体.测序后将MAGE-A3基因片段亚克隆至真核表达载体,构建成pIRES2-EGFP/MAGE-A3重组质粒.经脂质体转染至293T细胞株后,荧光定量PCR、Western-blotting法鉴定MAGE-A3基因的表达.结果:MAGE-A3基因正确克隆在plRES2-EGFP/MAGE-A3,测序结果与GenBank公布的MAGE-A3序列一致.转染293T细胞后能检测到MAGE-A3基因和蛋白的表达.结论:成功构建plRES2-EGFP/MAGE-A3真核表达质粒,并能在293T细胞中有效表达MAGE-A3蛋白,奠定了其作为DNA疫苗应用的基础.%Objective; To construct human melanoma antigen-encoding gene ( MAGE-A3 ) eukaryotic expression vector, and to identify and determine the expression in cell lines. Methods; MAGE-A3 gene was amplified by using polymerase chain reaction (PCR) and was linked to pMD18-T vector after harvesting PCR products via gel extraction. After confirmation by sequencing, the gene segments of MAGE-A3 were subcloned into eukaryotic expression vector for construction of pIRES2-EGFP/MAGE-A3, the recombinant plasmid. This was followed by transfection into the 293 T cell lines using liposomes for further determination of MAGE-A3 gene expression via fluorescent quantitative PCR and Western blotting. Results; MAGE-A3 gene was successfully cloned in the plasmid pIRES2-EGFP/MAGE-A3 , showing identical sequence of MAGE-A3 as compared with that reported in GenBank. The expression of MAGE-A3 genes and proteins were identified after transfection into the 293T cells. Conclusion;The successful construction of eukaryotic expression plasmid pIRES2-EGFP/MAGE-A3 was associated with effective expression of MAGE-A3 protein in 293T cell lines. This may offer grounds for the application of pIRES2-EGFP/MAGE-A3 as a DNA vaccine candidate in laryngocarcinoma immunotherapy.

  18. Cloning of rat glutamine synthetase gene and its expression in Hela-G cells%大鼠谷氨酰胺合成酶基因的克隆及其在Hela-G细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    孙伟峰; 刘春兴; 邹健

    2011-01-01

    Objective To clone rat glutamine synthetase(GS) gene and to express it in mammalian cells. Methods Rat GS cD NA was amplified by RT-PCR from RNA of rat cerebral cortex tissue. GS cDNA was inserted into eukaryotic expression vector pEGFP-N3. The recombinant expression vector was transiently transfected into Hela-G cells by LipofectamineTM 2000 reagent. The expression of GS in Hela-G cells was detected by immunocytochemistry. Results The sequence of the cloncd GS was confirmed by DNA sequencing. The Hela-G cells transfected with pEGFP-N3-GS could efficicntly express GS protein. Conclusion The cloning of rat GS gene and the construction of its eukaryotic expression vector are successful,which lavs the foundation for further investiga ting the role of GS in astrocytes.%目的 克隆大鼠谷氨酰胺合成酶(GS)基因,构建其真核表达载体,并观察其在Hela-G细胞中的表达. 方法 采用RT-PCR方法,以大鼠大脑皮层总RNA为模板,扩增GS基因,定向克隆到pEGFP-N3载体中.以LipofectamineTM2000试剂转染pEGFP-N3-GS表达载体至Hela-G细胞中进行瞬时真核表达.以免疫细胞化学方法鉴定GS的表达.结果 从大鼠大脑皮层组织中克隆到序列正确的GS全长编码序列.所构建的pEGFP-N3-GS质粒在Hela-G细胞中获得高效表达.结论 大鼠GS基因的克隆、真核表达载体的构建及在Hela-G中的表达获得成功.

  19. Construction of porcine CCK pDNA and its expression in COS-7 cells.

    Science.gov (United States)

    Bai, Jigang; Lü, Yi; Bai, Qiaoling

    2007-06-01

    CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamin 2000-mediated transfer method. The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected. And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells, which lays a foundation for further research on the relationship between CCK and tumor.

  20. Construction of Porcine CCK pDNA and Its Expression in COS-7 Cells

    Institute of Scientific and Technical Information of China (English)

    BAI Jigang; L(U) Yi; BAI Qiaoling

    2007-01-01

    CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamine TM2000-mediated transfer method.The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected.And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells,which lays a foundation for further research on the relationship between CCK and tumor.

  1. Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响%Effect of Ezrin gene knockdown and over-expression on invasion of glioma U87 cells

    Institute of Scientific and Technical Information of China (English)

    刘乃杰; 秦治刚; 孙利波; 金星一; 叶保国; 张金男; 朱庆三

    2013-01-01

    Objective To study the relationship between Ezrin gene and infiltrative growth of glioma through Ezrin gene knockdown and over-expression. Methods According to Ezrin gene sequence in GenBank, primers were designed using Prime Primer 5. 0 software, with which the gene fragment encoding CDS region of Ezrin gene was amplified from U87 cells and cloned into expression vector pEGFP-1. U87 cells were transfected with the constructed recombinant plasmid pEGFP-C 1/Ezrin as well as plasmids shRNA-EZrin-2 and pEGFP-C 1 in mediation of LipofectimineTM 2000 respectively, then deter-mined for expression of Ezrin protein by Western blot, and for migration by scarification test. Results The homologies of mRNAs of cloned Ezrin gene were 99% to those of homo sapiens ezrin (EZR) and transcript variant 1 reported in Gen-Bank. The homologies of amino acids encoding by the cloned gene was 99% to that of ezrin [Homo sapiens] (Sequence ID: ref-NP_003370. 2-, Length:586), with variations of S66P, K258R, P265L and K577R, while the opening read frame was correct. The relative expression level (1. 17) of Ezrin protein in U87 cells transfected with recombinant plasmid pEGFP-C1 /Ezrin was higher those transfected with shRNA-Ezrin-2 (0. 47) and pEGFP-C1 (0. 82). Scarification test showed migration of a small quantity of U87 cells transfected with shRNA-Ezrin-2 to the wound. However, in pEGFP-C1/ Ezrin transfection group, the wound was almost filled with cells. Conclusion Ezrin gene knockdown blocked , while the over-expression promoted the migration of U87 cells, indicating that Ezrin gene involved in the invasive growth of U87 cells.%目的 分析Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响,以探讨脑胶质瘤浸润性生长的机理.方法 从U87细胞中扩增Ezrin基因CDS区片段,克隆至表达载体pEGFP-C1中,构建Ezrin基因表达质粒pEGFP-C1/Ezrin.将pEGFP-C1/Ezrin、Ezrin基因shRNA质粒shRNA-Ezrin-2和pEGFP-C1以脂质体LipofectimineTM 2000

  2. Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens.

    Science.gov (United States)

    Guo, Tz-Chun; Johansson, Daniel X; Liljeström, Peter; Evensen, Øystein; Haugland, Øyvind

    2015-03-01

    A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5' end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3' end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.

  3. 透明质酸/壳聚糖/pEGFP纳米粒介导体外基因转染关节软骨细胞与滑膜细胞的比较%Hyaluronic acid/chitosan/pEGFP nanoparticles mediated gene transfection of articular chondrocytes and synoviocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    吕璐璐; 卢华定; 陆慧琼; 张富程; 赵慧清

    2012-01-01

    [目的]以透明质酸(HA)修饰的壳聚糖(CS)/质粒DNA(pDNA)纳米粒介导体外基因转染关节软骨细胞与滑膜细胞,以明确其作为非病毒基因载体治疗关节疾病的潜能.[方法]将HA修饰的CS与负载增强型绿色荧光蛋白基因(EGFP)的pDNA以复凝聚法制成纳米粒,以扫描电镜检测纳米粒形态;激光粒度仪测定其粒径、Ze-ta电位及分散度(PDl);凝胶电泳阻滞试验榆测HA/CS和pDNA的结合力及pDNA的释放;体外转染兔关节软骨细胞与滑膜细胞,以流式细胞仪及荧光显微镜检测转染效率.[结果]HA/CS/pDNA纳米粒多呈球形,粒径平均为(142.5±20.3)nm,表面Zeta电位平均为(25.99±8.48)mV,分散度平均为(0.283±0.089),可有效保护pDNA免受核酸酶的降解;通过调节pH值至7.5以上或加入壳聚糖酶可促使纳米粒中的pDNA释放;体外转染实验证明HA/CS/pDNA纳米粒能介导pEGFP转染软骨细胞和滑膜细胞并在细胞内表达绿色荧光蛋白,其对软骨细胞的转染能力较强,比裸pEGFP和CS/pEGFP纳米粒有更高的转染效率(P<0.05);但对滑膜细胞的基因转染效率较低,与CS/pEGFP纳米粒无明显差别(P>0.05).[结论]复凝聚法制备的HA/CS/pDNA纳米粒是一种有效的新型非病毒基因转染载体,在体外可介导基因转染关节软骨细胞和滑膜细胞,其转染效率具有明显的细胞依赖性.%[Objective] Hyaluronic acid ( HA) was used to modify ehitnaan/pDNA nanoparticles to produce HA/CS/pDNA nanoparticles as novel gene vectors. And to study HA/CS/pDNA nanoparticles mediated gene transfection of chondrocytes and synoviocytes in vitro, so as to ascertain them as potential non -viral gene vectors for the treatment of joint disease. [Methods ] The HA/CS/pDNA nanoparticles were prepared by a complex coacervation method with HA modified chitosan mixed with plasmid DNA (pDNA), which loaded enhanced green fluorescent protein (EGFP) gene. The morphology of nanoparticles was

  4. Experimental study of electroporation-mediated plasmid gene expression in skin and incisional wound%电穿孔介导质粒基因在皮肤和线性伤口中的表达

    Institute of Scientific and Technical Information of China (English)

    高振; 宋楠; 武晓莉; 曾谊林; 刘伟

    2008-01-01

    Objective To explore the feasibility of electroporation mediated gone transfer in rat incisional wound.Methods 12 Sprague-Dawley rat's dorsal skins were electroporated(800 voltages in amplitude with 6 square wsve pulses,each lasting 20 milliseconds with 200 millisecond interval)after injection of plasmid DNA(1 μg/μl,in 100 μl PBS)containing enhanced green fluorescence protein(EGFP)gene.Electroporated skins were incisiomdly wounded 24 hours after electroporation.Specimens were harvested at day 2,4,6,14,then EGFP expression in dennis wag observed and quantitatively analyzed with integrated optical density(IOD)followed by H&E staimng.Results Eleetroporation can mediate EGFP expression in epidermis,dermis and pannicuhs muscle.The expression level in dermis Wag the highest at dav 2(IOD=3.50±1.45)and disappeared at day 14.EGFP expression Wag not found in dennis if no electroporation apphed after plasmid injection(IOD=0).Conclusion Electroporation can mediate plagmid gone expression in ineisional wound efficiently and widely.%目的 研究在线性伤口中进行电穿孔基因转染的可行性.方法 于12只Sprague-Dawley大鼠背部皮肤内注射增强型绿色荧光蛋白(EGFP)质粒(1 μg/μl)后,在注射局部进行电穿孔(800 V/cm,6个方波,每个持续20ms,间隔为200ms).电穿孔后24 h在电穿孔部位做线性切口,于第2、4、6、14天收集标本.观察EGFP表达并计算其真皮内积分光密度(IOD).然后进行HE染色.结果 电穿孔使EGFP在表皮、真皮以及肉膜内表达,真皮内表达量在第2天最高(IOD=3.50±1.45),至第14天消失,而单纯质粒注射真皮内无表达(IOD=0).结论 电穿孔能够介导质粒在线性伤口中高效而广泛地表达.

  5. 11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity

    Science.gov (United States)

    Lv, Sai-qun; Ye, Zhen-long; Liu, Pin-yi; Huang, Yao; Li, Lin-fang; Liu, Hui; Zhu, Hai-li; Jin, Hua-jun; Qian, Qi-jun

    2017-01-01

    Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

  6. Construction of eukaryotic expression plasmid of Fasciola hepatica CatL gene and biological activity analysis of recombinant protein%肝片吸虫组织蛋白酶L真核表达载体构建及重组蛋白活性分析

    Institute of Scientific and Technical Information of China (English)

    闻晓波; 冉旭华; 王春仁; 宋佰芬; 魏晓曼; 李晓娟; 王密; 苗艳

    2013-01-01

    In this research we constructed eukaryotic expression plasmid expressing Fasciola hepalica cathepsin L-like proteases (CatL) and analyzed the immunogenicity of recombinant protein. CatL gene was amplified by PCR with the template of recombinant pET30a-FhCatL plasmid and cloned into pEGFP-Nl vector to construct recombinant plasmids pEGFP-Nl-CatL. The recombinant plasmid pEGFP-Nl-CatL was transfected into HeLa cells and fluorescent signal was detected by fluorescence microscope. The immunogenicity of recombinant protein identified by western blotting demonstrated that the recombinant protein specifically reacted with serum from goat infected by Fasciola hepalica and showed robust immunoreactivity. The eukaryotic expression plasmid may be a potential gene vaccine or diagnose preparations in further study.%目的 构建肝片吸虫组织蛋白酶L(CatL)的真核表达载体,研究重组蛋白的生物学活性.方法 以构建好的重组质粒pET30a-FhCatL为模板,利用PCR技术扩增肝片吸虫组织蛋白酶L基因(catL),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-N1-CatL,转染HeLa细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况.结果 重组质粒pEGFP-N1-CatL在HeLa细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应.结论 肝片吸虫CatL真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有免疫反应性,可做为分子疫苗的候选和诊断抗原进行进一步的研究.

  7. Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases.

    Science.gov (United States)

    Luo, Yongquan; Liu, Chengyu; Cerbini, Trevor; San, Hong; Lin, Yongshun; Chen, Guokai; Rao, Mahendra S; Zou, Jizhong

    2014-07-01

    Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a "safe harbor" locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken β-actin/rabbit β-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.

  8. Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells

    Institute of Scientific and Technical Information of China (English)

    Sheng-lin HUANG; Yi WU; Hai YU; Ping ZHANG; Xing-qian ZHANG; Lei YING; Han-fang ZHAO

    2006-01-01

    Aim: RNA interference (RNAi) has been proposed as a potential treatment for cancer, but the lack of cellular targets limits its use in cancer gene therapy. No current technology has achieved direct tumor-specific gene silencing using RNAi.In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase (hTERT) promoter; furthermore, we analyzed its inhibitive effect on Bcl-2 expression. Methods: The vectors containing a small hairpin RNA (shRNA) to target exogenous reporters [firefly luciferase and enhanced green fluorescent protein (EGFP)] and endogenous gene (Bcl-2)were constructed. Luciferase expression was determined by dual luciferase assay.Reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and fluorescence-activated cell sorting (FACS) were used to measure EGFP expression. Inhibition of Bcl-2 was evaluated by RT-PCR and Western blotting.Cell proliferation and viability were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. FACS was used to analyze the cell cycle distribution profile. Results: We showed that with the hTERT promoter directly driving shRNA transcription, expression of the exogenous reporters (LUC and EGFP) in tumor cells, but not normal cells, was specifically inhibited in vitro. The hTERT promoter-driven shRNA also depressed the expression of Bcl-2. Inhibition of Bcl-2 did not affect cell proliferation, but increased the chemosensitivity of HeLa cells to 5-fluorouracil. Conclusion: The present study describes an efficient RNAi system for gene silencing that is specific to tumor cells using the hTERT promoter. Suppression of Bcl-2 by using this system sensitized HeLa cells to 5-fluorouracil. This system may be useful for RNAi therapy.

  9. Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering

    Institute of Scientific and Technical Information of China (English)

    Ting Tan; Zhi-Qi Hu

    2013-01-01

    Objective:To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase(TERT) and to investigate the expression ofTERT in neonatal mouse hypodermal cells.Methods:The polymerase chain reaction(PCR)-amplifiedTERT gene was inserted into plasmid pLEGFP-N1.The positive clone was identified by restriction enzyme digestion and sequencing, then was transfected into packaging cells to produce retrovirus particles.Neonatal mouse hypodermal cells were infected with the virus to generate a stable cell line.TheTERT mRNA expression level, telomerase activity, and enhanced green fluorescent protein(EGFP) expression level were analyzed.Results:Retroviral vector pLEGFP-N1-TERT was constructed successfully, and a stable cell line of neonatal mouse hypodermal cells expressingEGFP was established.Western blot and immunohistochemical assay showed that the expression level ofTERT was significantly elevated in the neonatal mouse hypodermal cells.Conclusions:A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenousTERT gene in the neonatal mouse hypodermal cells.This protocol has potential applications for skin tissue engineering and cell transplantation therapy.

  10. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    Science.gov (United States)

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities.

  11. EGFP和CM-Dil示踪骨髓间充质干细胞构建组织工程骨的体内研究%The in vivo study of tissue engineered bone constructed with EGFP and CM-Dil labeled BMSCs

    Institute of Scientific and Technical Information of China (English)

    武京国; 谢方南; 马慧雨; 王黔; 曹谊林; 肖苒

    2012-01-01

    目的:应用增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)和CM-Dil标记技术,观察组织工程骨在体内形成过程中种子细胞的变化和转归.方法:分别用EGFP慢病毒表达和CM-Dil染料的方法标记比格犬骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs),MTT法检测标记细胞的体外增殖能力.BMSCs接种珊瑚支架体外成骨诱导7天后,将未标记组、EGFP组和CM-Dil组分别植入裸鼠背部皮下,空白支架作为阴性对照.术后4、8、12周取材,HE染色观察成骨情况,EGFP组采用GFP免疫组化、CM-Dil组冰冻切片荧光显微镜下示踪BMSCs在体内的变化.结果:两种标记技术能高效标记BMSCs,标记前后细胞的体外增殖无显著性差异(P>0.05).细胞-支架复合物植入体内12周后有新生骨形成,标记细胞数量随时间延长而逐渐减少,12周后仍显示有部分标记细胞存活.结论:EGFP和CM-Dil可用于示踪组织工程种子细胞,通过示踪说明BMSCs在体内组织工程骨成骨过程中发挥了重要作用.%Objective To observe the effect of BMSCs in fabricating tissue engineering bone in vivo using EGFP andCM-Dil labeling technology. Methods BMSCs isolated from Beagle Dogs were labeled using EGFP and CM -Dilseparately and the proliferation abilities were analyzed by MTT assay. BMSCs were seeded onto the coral scaffolds andcultured in the osteogenic medium for 7 days.Then the BMSCs/Coral constructs were implanted into the nude micessubcutaneously.The constructs were divided into three groups:Unlabeled group,EGFP group,CM -Dil group.Thespecimens were collected respectively at 4,8 and 12 weeks after implantation and tissue engineered bone wasevaluated by HE staining.The seeded BMSCs were traced by immunohistochemistry staining of GFP in the EGFP groupand directly observed in the frozen section under the fluorescence microscope in the CM-Dil. Results BMSCs werelabeled efficiently by both GFP and CM-Dil labeling technology and

  12. Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

    Indian Academy of Sciences (India)

    Nirpendra Singh; Atul Ranjan; Souvik Sur; Ramesh Chandra; Vibha Tandon

    2012-07-01

    HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80% and 70% respectively.

  13. Construction and identification of recombination expression vector Ksp-Cadherin-Gpx1-Kik1

    Institute of Scientific and Technical Information of China (English)

    Xie Liyi; Xue Wujun; Xiang Heli; Ma Sunkai

    2008-01-01

    Objective To construct and identify the Gpx1-Klk1 vector which contains kidney-specific promoter (Ksp-cadherin). Methods Through PCR amplification, the human Gpx1, Klk1, and Ksp-cadherin eDNA were obtained by taking Gpx1 cDNA, Klk1 eDNA, and Ksp-cadherin BAC as templates. After being testified, the PCR products were inserted into the expressive vector pIRES-EGFP step-by-step to produce a recombinant vector Ksp-cadherin-Gpx1-Klk1. This vector was examined by restriction enzyme digestion and sequence analysis. Results The recombinant expressive vector Ksp-cadherin-Gpx1-Klk1 was successfully constructed. Conclusion The construction of the recombinant vector Ksp-cadherin-Gpx1-Kik1 laid foundations for investigations in establishing transgenic animal models, the over-expression of Gpx1 and Kikl in mammal kidney, and gene therapy for ischemia-reperfnsion injury during kidney transplantation.

  14. Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells.

    Science.gov (United States)

    Krammer, Florian; Pontiller, Jens; Tauer, Christopher; Palmberger, Dieter; Maccani, Andreas; Baumann, Martina; Grabherr, Reingard

    2010-10-11

    Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.

  15. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

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    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  16. 靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响%Construction of Recombinant Lentiviral Vector of siRNA for Ang2 and the Effect of the Expression of Ang2 Gene in Malignant Melanoma Cells

    Institute of Scientific and Technical Information of China (English)

    刘照亮; 王彪; 郭国祥; 单秀英; 王美水; 庄福连; 蔡传书; 张明凤; 张彦定

    2011-01-01

    目的 构建携带促血管生成素2-小干扰RNA(Ang2-siRNA)慢病毒载体,观察其对恶性黑色素瘤细胞中Ang2基因表达的干扰作用.方法 将经XbaⅠ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒(pNL-EGFP)载体与pSilencer 1.0-U6启动子-促血管生成素2-小干扰RNA(pSilencer 1.0-U6-Ang2-siRNA)重组质粒连接,产生加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅰ(pNL-EGFP-U6-Ang2-Ⅰ)、加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅱ(pNL-EGFP-U6-Ang2-Ⅱ)慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒、水疱性口炎病毒G蛋白(pVSVG)包膜质粒和pHelper包装质粒共转染293T细胞,产生pNL-EGFP-U6-Ang2-Ⅰ、pNL-EGFP-U6-Ang2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Ang2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Ang2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.0×103/ml.实时荧光定量RT-PCR结果显示:Ang2-siRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Ang2基因的表达(P<0.05).结论 成功构建了Ang2-SiRNA慢病毒载体,体外研究显示Ang2-SiRNA慢病毒载体能抑制恶性黑色素瘤细胞中Ang2 mRNA的表达,为下一步进行裸鼠恶性黑色素瘤移植瘤生长的干预实验奠定基础,为肿瘤的基因治疗提供实验依据.%Objective To construct lentivectors carrying Ang2-small interfering RNA ( siR-NA ), which was used to inhibit Ang2 expression in malignant melanoma cells. Methods Plasmid pNL-EGFP and recombinant plasmid pSilencer 1. 0-U6-Ang2-siRNA were digested with Xbal and li-gated into a target lentiviral transfer plasmid of pNL-EGFP-U6-Ang2- I or pNL-EGFP-U6-Ang2- Ⅱ . Plasmids pNL-EGFP-U6-Ang2- I and pNL-EGFP-U6-Ang2- II were constructed successfully. Lentiviral

  17. Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    GAO Bo; SUN Huai-chang; SONG Cheng-yi; WANG Zhi-yue; CHEN Qin; SONG Hong-qin

    2005-01-01

    To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region.The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyleneimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis.The results showed that expression of pEGFP-N1 was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the ?-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

  18. In vitro expression of native H5 and N1 genes of avian influenza virus by using Green Fluorescent Protein as reporter

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    Risza Hartawan

    2011-10-01

    Full Text Available The hemagglutinin and neuraminidase are important immunogen of avian influenza virus that are suitable for recombinant experimentation. However, both genes have been experienced rapid mutation resulting in diverse variety of genotypes. Hence, gene expression in recombinant systems will be difficult to predict. The objective of the study was to examine expression level of H5 and N1 genes from a field isolate by cloning the genes into expression vector pEGFP-C1. Two clones respresenting fulllength of H5 and N1 gene in plasmid pEGFP-C1 were transfected into chicken embryo fibroblasts (CEF, rabbit kidney (RK13 and African green monkey kidney (VERO cells using Lipofectamine ‘Plus’ reagent. The experiment showed level of gene expression in the VERO cell was higher than in the RK13 and CEF cells. Observations using fluorescent microscopy and Western blotting revealed that the N1 gene was expressed better in all cells compared to the H5 gene.

  19. Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

    Science.gov (United States)

    Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

    2016-06-01

    Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

  20. Differential expression of human homeodomain TGIFLX in brain tumor cell lines.

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    Reza Raoofian

    2013-12-01

    Full Text Available Glioblastoma is the most common and the most lethal primary brain cancer. This malignancy is highly locally invasive, rarely metastatic and resistant to current therapies. Little is known about the distinct molecular biology of glioblastoma multiforme (GBM in terms of initiation and progression. So far, several molecular mechanisms have been suggested to implicate in GBM development. Homeodomain (HD transcription factors play central roles in the expression of genomic information in all known eukaryotes. The TGIFX homeobox gene was originally discovered in human adult testes. Our previous study showed implications of TGIFLX in prostate cancer and azoospermia, although the molecular mechanism by which TGIFLX acts is unknown. Moreover, studies reported that HD proteins are involved in normal and abnormal brain developments. We examined the expression pattern of TGIFLX in different human brain tumor cell lines including U87MG, A172, Daoy and 1321N1. Interestingly, real time RT-PCR and western blot analysis revealed a high level of TGIFLX expression in A172 cells but not in the other cell lines. We subsequently cloned the entire coding sequence of TGIFLX gene into the pEGFP-N1 vector, eukaryotic expression vector encoding eGFP, and transfected into the U-87 MG cell line. The TGIFLX-GFP expression was confirmed by real time RT-PCR and UV-microscopic analysis. Upon transfection into U87 cells, fusion protein TGIFLX-GFP was found to locate mainly in the nucleus. This is the first report to determine the nuclear localization of TGIFLX and evaluation of its expression level between different brain tumor cell lines. Our data also suggest that TGIFLX gene dysregulation could be involved in the pathogenesis of some human brain tumors.

  1. CCR7基因载体的构建及其在肺腺癌H157细胞的稳定表达%Construction of human CCR7 gene vector and its stable expression in lung adenocarcinoma H157 cells

    Institute of Scientific and Technical Information of China (English)

    郭学光

    2011-01-01

    Objective To construct expression vectors of human CCR7 gene and to obtain H157 cells that can express CCR7 stably. Methods CCR7 coding domain was amplified from lung adenocarcinoma patient using RT-PCR, directionally cloned into pEGFP-N1 plasmid. The recombinant vectors were transfected into H157 cell lines by DOTAP liposome and screened by G418 selective medium. The expression of CCR7 was verified by RT-PCR and flow cytometry. Results Correct construction of pEGFP-CCR7 was identified by methods of restriction enzyme analysis, PCR amplication and nucleotide-sequencing. CCR7 was found to be expressed in the transfected H157 cells with fluorescent microscopy, RT-PCR and flow cytometry. Conclusions The CCR7-expressing plasmid has been constructed successfully and CCR7 is expressed stably in human adenocarcinoma H157 cells.%目的 构建人CCR7基因真核表达质粒,获得稳定表达CCR7蛋白的H157细胞.方法 应用逆转录PCR法(RT-PCR)自人肺腺癌标本中扩增出CCR7编码区序列,定向克隆至载体pEGFP-N1中构建质粒pEGFP-CCR7,采用脂质体介导的基因转染技术将重组质粒DNA导入肺腺癌H157细胞中,加入G418对细胞进行筛选,获得稳定表达CCR7的细胞,并用荧光显微镜、RT-PCR和流式细胞术对CCR7的表达进行鉴定.结果 PCR、酶切及测序结果证明,重组质粒pEGFP-CCR7构建正确,荧光显微镜、RT-PCR及流式细胞术在稳定转染H157细胞中检测到人CCR7的表达.结论 成功构建人CCR7基因真核表达质粒并获得稳定表达人CCR7的H157细胞株.

  2. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

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    Margison Geoffrey P

    2006-03-01

    Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

  3. Potent and specific inhibition of SARS-CoV antigen expression by RNA interference

    Institute of Scientific and Technical Information of China (English)

    TAO Peng; ZHANG Jun; TANG Ni; ZHANG Bing-qiang; HE Tong-chuan; HUANG Ai-long

    2005-01-01

    Background Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. Methods The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.Results The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.Conclusions Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.

  4. Cloning and expression of chicken's BCLI0 gene%鸡BCLl0基因的克隆与表达

    Institute of Scientific and Technical Information of China (English)

    孙柏; 李燕丽; 张亚杰; 薄联锋; 郜原; 张德礼

    2011-01-01

    To detect the existence of BCL10 gene in chickens and research how it expressed in E. coli and vero cells.A full-length cDNA of 959 bp was acquired by silico cloning and the ORF of 735 bp contained in the cDNA was cloned by RT-PCR. The chickens BCL10 gene was cloned to the prokaryotic expression vector pET-32a and eukaryotic expression vector pEGFP-C1. The expression product of prokaryotic expression vector pET-BCL10 was detected by SDS-PAGE. Eukaryotic expression vector pEGFP-BCL10 was transferred to vero cell line by lipofectin 2000 and 6GFP was detected by Western blotting. The results showed that the sequence obtained by silico cloning was the same as that obtained by sequencing. Prokaryotic expression vector and eukaryotic expression vector were both successfully constructed by sequencing. pET-BCL10 was expressed in E. coli confirmed by SDS-PAGE. BCL10 gene was successfully expressed in vero cells by GFP marker and Western blotting, The results indicated that the BCL10 gene definitely exists in chickens, which could be successfully expressed in E. coli and vero cells.%通过电子克隆手段,克隆到大小为959 bp的cDNA序列.然后从鸡的脾脏提取RNA,用RT-PCR方法扩增出目的片段.将该片段连接到原核表达栽体pET-32a和真核表达载体pEGFP-C1中,采用SDS-PAGE检测原核表达载体pET-BCL10在大肠杆菌中的表达,脂质体转染法将真核表达载体pEGFP-BCL10转入vero细胞,Western blotting检测其在细胞中的表达.结果显示,PCR扩增到735 bp的DNA片段,原核表达载体和真核表达栽体经双酶切和核酸测序表明载体构建成功.SDS-PAGE电泳检测到pET-BCL10重组质粒在大肠杆菌中表达.western blotting检测到pEGFP-BCL10重组质粒在veto细胞中表达.结论表明,BCLl0基因在鸡体内存在,该基因的重组质粒能在大肠杆菌和vero细胞中表达.

  5. PLSCR1真核表达载体的构建及细胞内定位分析%Construction of the eukaryotic expression vector of PLSCR1 and analysis of its intracellular localization

    Institute of Scientific and Technical Information of China (English)

    王远; 陈英; 张学清; 陈忠民; 田喜凤; 陈晶

    2015-01-01

    分别构建带有myc标签和GFP荧光蛋白的磷脂爬行酶1(PLSCR1)真核表达载体,获得两个融合表达载体,并转入HEK293细胞观察表达情况及细胞内定位,为研究PLSCR1的定位与功能的关系奠定基础。以本实验室保存的Hela cDNA文库为模板,采用PCR技术扩增PLSCR1编码序列,将其分别插入pCMV-Myc-N和pEGFP-C1载体, Western blotting检测其在HEK293中的表达,采用激光共聚焦观察pEGFP-C1融合表达载体在HEK293细胞中定位。通过DNA序列分析,证实了成功构建了PLSCR1真核表达载体,并能在HEK293细胞中实现基因的过表达。成功构建PLSCR1真核表达载体,为进一步研究其功能奠定了基础。%Two eukaryotic expression vectors of phospholipid scramblase 1(PLSCR1)with myc-tag or Green Fluorescent Protein (GFP), were constructed to obtain two fusion expression vectors, which were transfected to HEK293 cell, to observe the expression and cellular localization. The results would lay a foundation for the study of PLSCR1 gene localization and functional relationships. Hela cDNA library preserved in our laboratory was used as template, the PLSCR1 coding sequence was amplified by PCR and respectively inserted into the vector pCMV-Myc-N and pEGFP-C1. The epression was detected in HEK293 by Western blotting and localization of pEGFP-C1 fusion expression vector in HEK293 cells by laser scanning confocal microscopy. As results, the eukaryotic expression vector of PLSCR1 was successfully constructed by the DNA sequence analysis, and over expressed genes in HEK293 cells. It makes good foundation for further study of functions by successfully constructing eukaryotic expression vector of PLSCR1.

  6. Construction of eukaryotic expression vector system for expression of VP1 gene of encephalomyocarditis virus%脑心肌炎病毒VP1基因真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    凡静静; 冯若飞; 杨妍梅; 张海霞; 李向茸; 王丹; 谢晶莹; 马忠仁

    2013-01-01

    The objective of this study was to construct a eukaryotic expression vector harboring VP1 gene of encephalomyocarditis virus(EMCV) ,and to express the VP1 gene in CHO cells. VP1 gene segment of EMCV was amplified by RT-PCR,and then cloned into the pEGFP-Cl vector to construct eukaryotic expression vector pEGFP-C1-VP1. The constructed plasmid was then transfected into the CHO cell via Lipo-fectamineTM 2000 Reagent. Transcription of VP1 gene in the CHO cell was tested via RT-PCR,and localization and reactinogenicity of VP1 protein in the CHO cell were detected by using immunohistochemistry and Western-blot,respectively. Enzyme digestion of PCR products and sequencing showed that the recombinant plasmid pEGFP-Cl-VPl was successfully constructed. RT-PCR result showed that the VP1 gene was tran-scripted in the CHO cell. Immunohistochemistry result showed that the expressed VP1 protein was mainly distributed in the membrane of CHO cells, and a little in cytoplasm. Western-blot showed that the expressed VP1 protein could bind to rabbit anti-EMCV antibody,indicating the immunoreactivity of recombinant VP1 protein.%为构建脑心肌炎病毒(EMCV) VP1基因的真核表达载体,并在CHO细胞中进行表达,通过RT-PCR扩增目的基因,酶切连接后将VP1基因克隆入真核表达载体pEGFP-C1中,通过脂质体法转染重组质粒pEGFP-C1-VP1,提取CHO细胞的总RNA,用RT-PCR检测目的基因的转录情况,并利用免疫组织化学和Western-blot分析检测目的基因表达情况、VP1蛋白在细胞中的定位及其抗原性.PCR、酶切验证及测序结果证实,重组质粒构建成功,绿色荧光蛋白正常表达.提取试验组细胞的总RNA,进行RT-PCR,在900 bp处扩增出了目的条带.免疫组织化学结果显示,VP1主要分布在CHO细胞的细胞膜中,少量存在于胞浆中.Western-blot分析结果证实,VP1能与兔抗EMCV抗体特异性结合.表明VP1蛋白在CHO细胞中表达良好,表达的蛋白具有反应原性.

  7. Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Zhen-Liang Qu; Sheng-Quan Zou; Nai-Qiang Cui; Xian-Zhong Wu; Ming-Fang Qin; Di Kong; Zhen-Li Zhou

    2005-01-01

    AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency.Cells were harvested and total RNA was extracted using TRIzol() reagent. The expression of hTERT mRNA in HepG2and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector.Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939cells only when transfected with HBx gene.CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

  8. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

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    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  9. Cloning and Expression Characteristics of the Pig Stra8 Gene

    Directory of Open Access Journals (Sweden)

    Xiaoyan Wang

    2014-07-01

    Full Text Available Stra8 (Stimulated by Retinoic Acid 8 is considered a meiotic gatekeeper gene. Using reverse transcriptase PCR and rapid amplification of cDNA ends (RACE, the complete sequence of the pig Stra8 gene was cloned. Bioinformatics analyses of this sequence were performed. Using semi-quantitative methods, the expression characteristics of Stra8 in Testis, cauda epididymis, body epididymis, caput epididymis, seminal vesicles, prostate gland, Cowper’s gland, heart, liver, spleen, lung, kidney, stomach, hypothalamus, pituitary gland, cerebrum, cerebellum, and hippocampus of adult Meishan boar and sow tissues were examined. The expression pattern in the testis of 2-, 30-, 60-, 90-, and 150-day old Meishan boars were analyzed using real-time PCR. We constructed a eukaryotic expression vector for the Stra8 gene and used it to transfect NIH-3T3 cells and third generation pig spermatogonial stem cells (SSCs cultured in vitro. Testes weight and sperm count in the cauda epididymis were evaluated at various time points. The results showed that the length of the pig Stra8 gene cDNA was 1444 bp encoding 366 amino acids with one typical helix-loop-helix (HLH domain. It is testes-specific expression. Expression was first detected in boar testis starting at day 2, and its expression significantly (p < 0.05 increased with age and body weight. When NIH-3T3 cells and pig SSCs were transfected with the eukaryotic expression vector EGFP (enhanced green fluorescent protein-N1-pStra8, it was expressed in the cytoplasm of NIH-3T3 cells. However, in SSCs, Stra8 was expressed predominantly in cytoplasm and few in nucleus. Our data suggest that perhaps Stra8 acts as a transcription factor to initiate meiosis in young boar.

  10. Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

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    Mehdi Sharifi Tabar

    2015-10-01

    Full Text Available Objective: Genetic modification of human embryonic stem cells (hESCs is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI into the target cell line, however, there are few reports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for transient expression of green fluorescent protein in two genetically different hESC lines, Royan H5 (XX and Royan H6 (XY, as well as human foreskin fibroblasts (hFF. For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively, were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after transfection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs was conducted to confirm stable integration of the transgene. Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation efficiency revealed that the vector concentrations from 20-60 μg and the density of the subjected cells (5×105 and 1×106 cells were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. Conclusion

  11. Expression of Kruppel-like factor KLF4 in mouse hair follicle stem cells contributes to cutaneous wound healing.

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    Juan Li

    Full Text Available BACKGROUND: Kruppel-like factor KLF4 is a transcription factor critical for the establishment of the barrier function of the skin. Its function in stem cell biology has been recently recognized. Previous studies have revealed that hair follicle stem cells contribute to cutaneous wound healing. However, expression of KLF4 in hair follicle stem cells and the importance of such expression in cutaneous wound healing have not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative real time polymerase chain reaction (RT-PCR analysis showed higher KLF4 expression in hair follicle stem cell-enriched mouse skin keratinocytes than that in control keratinocytes. We generated KLF4 promoter-driven enhanced green fluorescence protein (KLF4/EGFP transgenic mice and tamoxifen-inducible KLF4 knockout mice by crossing KLF4 promoter-driven Cre recombinase fused with tamoxifen-inducible estrogen receptor (KLF4/CreER™ transgenic mice with KLF4(flox mice. KLF4/EGFP cells purified from dorsal skin keratinocytes of KLF4/EGFP transgenic mice were co-localized with 5-bromo-2'-deoxyuridine (BrdU-label retaining cells by flow cytometric analysis and immunohistochemistry. Lineage tracing was performed in the context of cutaneous wound healing, using KLF4/CreER™ and Rosa26RLacZ double transgenic mice, to examine the involvement of KLF4 in wound healing. We found that KLF4 expressing cells were likely derived from bulge stem cells. In addition, KLF4 expressing multipotent cells migrated to the wound and contributed to the wound healing. After knocking out KLF4 by tamoxifen induction of KLF4/CreER™ and KLF4(flox double transgenic mice, we found that the population of bulge stem cell-enriched population was decreased, which was accompanied by significantly delayed cutaneous wound healing. Consistently, KLF4 knockdown by KLF4-specific small hairpin RNA in human A431 epidermoid carcinoma cells decreased the stem cell population and was accompanied by compromised

  12. A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei

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    Subramanian, Venkataramanan; Schuster, Logan A.; Moore, Kyle T.; Taylor, Larry E.; Baker, John O.; Vander Wall, Todd A.; Linger, Jeffrey G.; Himmel, Michael E.; Decker, Stephen R.

    2017-02-06

    The industrial workhorse fungus, Trichoderma reesei, is typically exploited for its ability to produce cellulase enzymes, whereas use of this fungus for over-expression of other proteins (homologous and heterologous) is still very limited. Identifying transformants expressing target protein is a tedious task due to low transformation efficiency, combined with highly variable expression levels between transformants. Routine methods for identification include PCR-based analysis, western blotting, or crude activity screening, all of which are time-consuming techniques. To simplify this screening, we have adapted the 2A peptide system from the foot-and-mouth disease virus (FMDV) to T. reesei to express a readily screenable marker protein that is co-translated with a target protein. The 2A peptide sequence allows multiple independent genes to be transcribed as a single mRNA. Upon translation, the 2A peptide sequence causes a 'ribosomal skip' generating two (or more) independent gene products. When the 2A peptide is translated, the 'skip' occurs between its two C-terminal amino acids (glycine and proline), resulting in the addition of extra amino acids on the C terminus of the upstream protein and a single proline addition to the N terminus of the downstream protein. To test this approach, we have cloned two heterologous proteins on either side of a modified 2A peptide, a secreted cellobiohydrolase enzyme (Cel7A from Penicillium funiculosum) as our target protein, and an intracellular enhanced green fluorescent protein (eGFP) as our marker protein. Using straightforward monitoring of eGFP expression, we have shown that we can efficiently monitor the expression of the target Cel7A protein.

  13. Expression of transgenes targeted to the Gt(ROSA26Sor locus is orientation dependent.

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    Douglas Strathdee

    Full Text Available BACKGROUND: Targeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene. METHODOLOGY: In order to develop a reliable system for reproducibly expressing transgenes it was decided to insert constructs at the Gt(ROSA26Sor locus. A cytomegalovirus (CMV promoter was used to drive expression of the Tetracycline (tet transcriptional activator, rtTA2(s-M2, and test the effectiveness of using the ROSA26 locus to allow transgene expression. The tet operator construct was inserted into one allele of ROSA26 and a tet responder construct controlling expression of EGFP was inserted into the other allele. CONCLUSIONS: Expression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at the locus. Additionally we have been able to determine the optimal orientation for transgene expression at the ROSA26 locus.

  14. 携带DREAM基因小分子干扰RNA重组质粒的构建%Construction of recombinant DREAM-targeting small interfering RNA expressing plasmids

    Institute of Scientific and Technical Information of China (English)

    陈敏; 项红兵; 田玉科

    2011-01-01

    BACKGROUND:DREAM is a multi-functional protein, which combine with different target proteins at different sites in cells. In vitro cultivate tests and animal experiments confirmed that DREAM is involving in onset mechanism of many different diseases.OBJECTIVE:To construct recombinant DREAM-targeting small interfering RNA (siRNA) expressing plasmids.METHODS:Oligonucleotide containing the small hairpin of DREAM was designed and synthesized, which was inserted into the pDC316-EGFP-U6 plasmid double digested by BamH Ⅰ and Hind Ⅲ. The liation product was transformed competence E.coli DH5α. Positive clones were identified by PCR and sequencing.RESULTS AND CONCLUSION:The result of PCR and gene sequencing confirmed that the pDC316-EGFP-DREAMshRNA-U6 recombinant plasmid with 473 bp had been constructed successfully.%背景:DREAM是一种多功能蛋白,在细胞中不同位置与不同靶蛋白结合,体外细胞培养和动物实验均证明DREAM参与了许多疾病的发病机制.目的:构建携带DREAM基因的小分子干扰RNA重组质粒.方法:设计并合成shRNA对应的两条互补的寡核苷酸链,pDC316-EGFP-U6质粒经BamHⅠ和HindⅢ双酶切与退火后的寡核苷酸连接,转化感受态E.coli DH5α,获得阳性克隆进行PCR和测序鉴定.结果与结论:经PCR、酶切及测序证实,重组质粒pDC316-EGFP-DREAM-shRNA-U6片段大小为473 bp,其中插入的片断序列和位点与预期完全一致,说明pDC316-EGFP-DREAM-shRNA-U6重组质粒构建成功.

  15. Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

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    Saori Tsuji

    Full Text Available Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

  16. Establishment of A Malignant Pleural Effusion Mouse Model with Lewis Lung 
Carcinoma Cell Lines Expressing Enhanced Green Fluorescent Protein

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    Xingqun MA

    2012-06-01

    Full Text Available Background and objective Malignant pleural effusion (MPE is a poor prognosis factor in patients with advanced lung cancer. The aim of this study is to establish a mouse model of MPE using Lewis lung carcinoma (LLC cell lines expressing enhanced green fluorescent protein (EGFP. Methods The mouse model was created by injecting LLC-EGFP cells directly into the pleural cavity of mice that were sacrificed periodically. The dynamic growth and metastasis of tumor cells were screened using in vivo fluorescence imaging. The remaining mice were subjected to transverse computed tomography (CT imaging periodically to analyze the formation rate of pleural effusion. The survival rate and tumor metastasis were also observed. Pleural fluid was gently aspirated using a 1 mL syringe and its volume was measured. When two or more mice bore pleural effusion at the same time, we calculated the average volume. The correlation of pleural effusion with the integrated optical density (IOD were analyzed. Results Four days after the inoculation of LLC-EGFP cells, green fluorescence was observed by opening the chest wall. The tumor formation rate was 100%, and the IOD gradually increased after inoculation. The metastasis sites were mediastinal, and the hilar lymph nodes were contralateral pleural as well as pericardial. The metastasis rates were 87%, 73% and 20%, respectively. The CT scan revealed that the formation rates of pleural effusion on days 7, 14 and 21 were 13%, 46% and 53%, respectively. The average volume of pleural effusion increased obviously on day 10 and peaked on day 16 with a value of 0.5 mL. The mean survival time of nude mice was 28.8 days. The volume of pleural effusion and IOD were significantly correlated (r=0.91, P<0.000,1. Conclusion A mouse model of lung cancer malignant pleural effusion was successfully established by injecting LLC lines expressing EGFP into the pleural cavity under a microscope. The model can enable dynamic observations of the

  17. Recombinant protein expression by targeting pre-selected chromosomal loci

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    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

  18. Functional Characterization of the Mannitol Promoter of Pseudomonas fluorescens DSM 50106 and Its Application for a Mannitol-Inducible Expression System for Pseudomonas putida KT2440.

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    Jana Hoffmann

    Full Text Available A new pBBR1MCS-2-derived vector containing the Pseudomonas fluorescens DSM10506 mannitol promoter PmtlE and mtlR encoding its AraC/XylS type transcriptional activator was constructed and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with Pseudomonas putida and eGFP as reporter gene. The new vector was applied for functional characterization of PmtlE. Identification of the DNA binding site of MtlR was achieved by in vivo eGFP measurement with PmtlE wild type and mutants thereof. Moreover, purified MtlR was applied for detailed in vitro investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to PmtlE as a dimer. The proposed DNA binding site of MtlR is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of PmtlE. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase.

  19. Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells.

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    Gilbert, Leona; Välilehto, Outi; Kirjavainen, Sanna; Tikka, Päivi J; Mellett, Mark; Käpylä, Pirjo; Oker-Blom, Christian; Vuento, Matti

    2005-01-17

    A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

  20. Construction of eukaryotic expression vector and expression of stage-specific gene T314 from newborn larvae of Trichinella spiralis%旋毛虫新生幼虫期特异性T314基因真核表达质粒的构建及表达

    Institute of Scientific and Technical Information of China (English)

    于建立; 白雪; 吴秀萍; 刘明远

    2012-01-01

    目的 构建旋毛虫(Trichinella spirais)新生幼虫期特异性T314基因真核表达质粒,并在BHK细胞中进行表达.方法 从旋毛虫新生幼虫RNA中通过RT-PCR技术扩增无信号肽T314全长基因,定向克隆至真核表达载体pEGFP-N1中,构建重组真核表达质粒T314-pEGFP-N1,脂质体法转染BHK细胞,荧光显微镜观察EGFP的表达,Western blot检测T314融合蛋白的表达.结果 重组真核表达质粒T314-pEGFP-N1经双酶切及测序证实构建正确;转染的BHK细胞48 h转染效率最高;表达的T314融合蛋白可与旋毛虫感染的猪血清发生特异性反应.结论 已成功构建了T314基因重组真核表达质粒T314-pEGFP-N1,并在BHK细胞中融合表达,为进一步研究旋毛虫包囊形成机制奠定了基础.%Objective To construct a eukaryotic expression vector for stage-specific gene T314 from newborn larvae of Trichinella spiralis and express in BHK cells. Methods Signal peptide-free full-length T314 gene was amplified from RNA of newborn larvae of T. Spiralis by RT-PCR and cloned into eukaryotic expression vector Pegfp-Nl. BHK cells were transfected with the constructed recombinant plasmid T314-Pegfp-Nl in mediation of Hposome, then observed for expression of EGFP by fluorescent microscopy, and for that of T314 fusion protein by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid T314-Pegfp-Nl was constructed correctly. The transfection efficacy of BHK cells reached the maximum 48 h after transfection. The expressed T314 fusion protein showed specific reaction with porcine serum infected with T. Spiralis. Conclusion The recombinant eukaryotic expression vector T314-Pegfp-Nl for T314 gene was successfully constructed, and fusion protein was expressed in BHK cells, which laid a foundation of further study on mechanism of nurse cell formation of T. Spiralis.

  1. G-patch domain containing 2, a gene highly expressed in testes, inhibits nuclear factor-κB and cell proliferation.

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    Hu, Fen; Gou, Lixia; Liu, Qing; Zhang, Wendian; Luo, Mengmeng; Zhang, Xiujun

    2015-02-01

    G-patch domain containing 2 (GPATC2), a human gene that is highly expressed in the testes, was implicated as a novel cancer/testis antigen. The present study investigated GPATC2 expression in a number of human cell lines and rat tissues, and its potential biological function in 293T cells. Semi‑quantitative reverse transcription-polymerase chain reaction analysis showed that GPATC2 was widely expressed in 15 human cell lines (representing different lineages) and in 11 different rat tissues, and that the GPATC2 mRNA relative expression level was significantly higher in the testis than it was in other tissues. 293T cells were transiently transfected with GPATC2-p enhanced green fluorescent protein (EGFP)‑N1 or GPATC2-pEGFP-C3 and the nuclei were stained with 4',6'‑diamidino‑2‑phenylindole. The results showed that GPATC2 is predominantly expressed in the nucleus of 293T cells. Overexpression of GPATC2 may inhibit transcription of the NF-κB reporter gene. The role of GPATC2 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that over‑expression of GPATC2 in 293T cells significantly inhibited cell proliferation by decreasing the number of cells in S phase. By contrast, GPATC2 knockdown by RNA interference exhibited the opposite effect, suggesting that GPATC2 may be involved in inhibiting G1-S phase transition in 293T cells. In conclusion, these results provide novel insight into the breadth of expression of GPATC2 and its role in cell proliferation.

  2. Sphingosine-1-phosphate receptor-1 (S1P1) is expressed by lymphocytes, dendritic cells, and endothelium and modulated during inflammatory bowel disease

    Science.gov (United States)

    Karuppuchamy, Thangaraj; Behrens, En-hui; González-Cabrera, Pedro; Sarkisyan, Gor; Gima, Lauren; Boyer, Joshua D.; Bamias, Giorgos; Jedlicka, Paul; Veny, Marisol; Clark, David; Peach, Robert; Scott, Fiona; Rosen, Hugh; Rivera-Nieves, Jesús

    2016-01-01

    The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of DSS, after colitis induced by transfer of CD4+CD45RBhi cells and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with IBD were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T cell velocity and induced S1P1 degradation and retention of naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function. PMID:27049060

  3. Generation and characterization of transgenic mice expressing mitochondrial targeted red fluorescent protein selectively in neurons: modeling mitochondriopathy in excitotoxicity and amyotrophic lateral sclerosis

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    Wang Yi

    2011-11-01

    Full Text Available Abstract Background Mitochondria have roles or appear to have roles in the pathogenesis of several chronic age-related and acute neurological disorders, including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Parkinson's disease, and cerebral ischemia, and could be critical targets for development of rational mechanism-based, disease-modifying therapeutics for treating these disorders effectively. A deeper understanding of neural tissue mitochondria pathobiologies as definitive mediators of neural injury, disease, and cell death merits further study, and the development of additional tools to study neural mitochondria will help achieve this unmet need. Results We created transgenic mice that express the coral (Discosoma sp. red fluorescent protein DsRed2 specifically in mitochondria of neurons using a construct engineered with a Thy1 promoter, specific for neuron expression, to drive expression of a fusion protein of DsRed2 with a mitochondrial targeting sequence. The biochemical and histological characterization of these mice shows the expression of mitochondrial-targeted DsRed2 to be specific for mitochondria and concentrated in distinct CNS regions, including cerebral cortex, hippocampus, thalamus, brainstem, and spinal cord. Red fluorescent mitochondria were visualized in cerebral cortical and hippocampal pyramidal neurons, ventrobasal thalamic neurons, subthalamic neurons, and spinal motor neurons. For the purpose of proof of principle application, these mice were used in excitotoxicity paradigms and double transgenic mice were generated by crossing Thy1-mitoDsRed2 mice with transgenic mice expressing enhanced-GFP (eGFP under the control of the Hlxb9 promoter that drives eGFP expression specifically in motor neurons and by crossing Thy1-mitoDsRed2 mice to amyotrophic lateral sclerosis (ALS mice expressing human mutant superoxide dismutase-1. Conclusions These novel transgenic mice will be a useful tool for better understanding

  4. RNA-Eluting Surfaces for the Modulation of Gene Expression as A Novel Stent Concept

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    Olivia Koenig

    2017-02-01

    Full Text Available Presently, a new era of drug-eluting stents is continuing to improve late adverse effects such as thrombosis after coronary stent implantation in atherosclerotic vessels. The application of gene expression–modulating stents releasing specific small interfering RNAs (siRNAs or messenger RNAs (mRNAs to the vascular wall might have the potential to improve the regeneration of the vessel wall and to inhibit adverse effects as a new promising therapeutic strategy. Different poly (lactic-co-glycolic acid (PLGA resomers for their ability as an siRNA delivery carrier against intercellular adhesion molecule (ICAM-1 with a depot effect were tested. Biodegradability, hemocompatibility, and high cell viability were found in all PLGAs. We generated PLGA coatings with incorporated siRNA that were able to transfect EA.hy926 and human vascular endothelial cells. Transfected EA.hy926 showed significant siICAM-1 knockdown. Furthermore, co-transfection of siRNA and enhanced green fluorescent protein (eGFP mRNA led to the expression of eGFP as well as to the siRNA transfection. Using our PLGA and siRNA multilayers, we reached high transfection efficiencies in EA.hy926 cells until day six and long-lasting transfection until day 20. Our results indicate that siRNA and mRNA nanoparticles incorporated in PLGA films have the potential for the modulation of gene expression after stent implantation to achieve accelerated regeneration of endothelial cells and to reduce the risk of restenosis.

  5. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

    Science.gov (United States)

    Feng, Xiujing; Cao, Shaoxian; Wang, Huili; Meng, Chunhua; Li, Jingxin; Jiang, Jin; Qian, Yong; Su, Lei; He, Qiang; Zhang, Qingxiao

    2015-02-01

    Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

  6. Identification and Characterization of a Rat Novel Gene RSEP4 Expressed Specifically in Central Nervous System

    Institute of Scientific and Technical Information of China (English)

    Xi-Dao WANG; Ling-Wei KONG; Zhi-Qin XIE; Yu-Qiu ZHANG; Zhi-Xin LIN; Zhi-Qi ZHAO; Lei YU; Nai-He JING

    2004-01-01

    The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length ofRSEP4 cDNA is 2006 bp, with a 501 nucleotide open reading frame (ORF) that encodes a 167 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1, CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

  7. Regulation of endothelial-specific transgene expression by the LacI repressor protein in vivo.

    Directory of Open Access Journals (Sweden)

    Susan K Morton

    Full Text Available Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2 with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R protein.

  8. Pbx-dependent regulation of lbx gene expression in developing zebrafish embryos.

    Science.gov (United States)

    Lukowski, Chris M; Drummond, Danna Lynne; Waskiewicz, Andrew J

    2011-12-01

    Ladybird (Lbx) homeodomain transcription factors function in neural and muscle development--roles conserved from Drosophila to vertebrates. Lbx expression in mice specifies neural cell types, including dorsally located interneurons and association neurons, within the neural tube. Little, however, is known about the regulation of vertebrate lbx family genes. Here we describe the expression pattern of three zebrafish ladybird genes via mRNA in situ hybridization. Zebrafish lbx genes are expressed in distinct but overlapping regions within the developing neural tube, with strong expression within the hindbrain and spinal cord. The Hox family of transcription factors, in cooperation with cofactors such as Pbx and Meis, regulate hindbrain segmentation during embryogenesis. We have identified a novel regulatory interaction in which lbx1 genes are strongly downregulated in Pbx-depleted embryos. Further, we have produced a transgenic zebrafish line expressing dTomato and EGFP under the control of an lbx1b enhancer--a useful tool to acertain neuron location, migration, and morphology. Using this transgenic strain, we have identified a minimal neural lbx1b enhancer that contains key regulatory elements for expression of this transcription factor.

  9. H5N1禽流感病毒NS1蛋白与干扰素诱导蛋白10表达的相关性研究%Influence of avian influenza virus NS1 protein on the expression of IP-10 in BEAS-2B cells

    Institute of Scientific and Technical Information of China (English)

    贾晓俊; 周剑芳; 王晶钰; 董婕; 薄洪; 李梓; 李魁彪; 蓝雨; 舒跃龙

    2008-01-01

    目的 研究高致病性禽流感(HPAI)H5N1病毒NS1蛋白对干扰素诱导蛋白10(IP-10)的影响.方法 分别将禽流感病毒A/Anhui/1/2005(H5N1)的NS1基因、插入80-84位缺失氨基酸的NS1突变基因及流感病毒A/Puerto Rico/8/1934(H1N1)的NS1基因克隆至真核表达载体pEGFP-N1,转染人支气管上皮细胞BEAS-2B,流式细胞仪检测转染细胞内IP-10的表达情况.结果 与pEGFP-N1对照组相比,三种NS1蛋白均能下调BEAS-2B细胞IP-10的表达(P0.01).结论 A/Anhui/1/2005(H5N1)禽流感病毒单一NS1蛋白能够抑制BEAS-2B细胞IP-10表达,但这并不能完全阐明其与病毒致病性之间的关系.%Objective To investigate the influence of avian influenza virus (AIV) NS1 protein on the expression of interferon-inducible protein 10 (IP-10). Methods NS1 gene from virus A/Anhui/1/2005 (H5N1),NS1 gene inserted with 80-84 amino acids from virus A/Anhui/1/2005(H5N1)and NS1 gene from virus A/Puerto Rico/8/1934 (H1N1) were cloned into the eukaryotic expression vector pEGFP-N1, and transected into BEAS-2Bcells, IP-10 expression level in transected cells was detected by flow cytometry. Results Compared with the control group pEGFP-N1, Expression of these three different NS1 genes can down-regulate the expression of IP-10in BEAS-2B cells, but there is no significant difference as to the lower level among them. Conclusion NS1protein of A/Anhui/1/2005(H5N1) can down-regulate the expression level of IP-10, but this may not clarify its relationship with the virulence of AIV.

  10. Study on Up-Regulated Expressions of Anti-HIV Genes by vMIP-Ⅰin Jurkat Cell%vMIP-Ⅰ激活Jurkat细胞抗-HIV基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    尹小菲; 陈彬; 谭晓华; 罗燕; 杨磊

    2012-01-01

    In this study,through the construction of eukaryotic expressive vector,the recombinant plasmids pEGFP-N3-vMIP-Ⅰ was transfected into Jurkat cells by electroporation.Then,by QRT-PCR technique,we detected the expression levels of anti-HIV genes:CCL5,APOBEC3F,MX1 in Jurkat cells which influenced by vMIP-Ⅰ gene and explored the mechanisms of vMIP-Ⅰ against HIV infection.The results he recombinant plasmids of pEGFP-N3-vMIP-Ⅰ was successfully constructed and electroporation transfection efficiency reached about 40%.In comparison with non-transfected gourp,the transfected vMIP-Ⅰ group can increase CCL5,A3G,A3F,and MX1 of Jurkat cells by 7.37,1.58,2.42 and 2.064 times seperately.The results suggest vMIP-Ⅰ can activate expressions of some relative anti-HIV genes in Jurkat cells,which probably is one of the mechanisms for anti-HIV infection.%本研究通过构建真核表达载体pEGFP-N3-vMIP-Ⅰ,电穿孔法将其转染至Jurkat细胞,荧光定量PCR检测vMIP-Ⅰ基因对Jurkat细胞内CCL5、APOBEC3G、APOBEC3F、等抗-HIV基因表达水平的影响,从而探讨vMIP-Ⅰ抗HIV感染的机制。结果显示:成功构建了pEGFP-N3-vMIP-Ⅰ载体,电穿孔转染效率达到40%左右,与转染空载体组相比,vMIP-Ⅰ转染组的Jurkat细胞内CCL5、A3G、A3F和MX1分别上调7.37倍、1.58倍、2.42倍和2.06倍。研究结果表明:vMIP-Ⅰ基因可激活Jurkat细胞内一些抗HIV相关基因的表达,这可能是vMIP-Ⅰ基因抗HIV感染的机制之一。

  11. Roles of extracellular signal-regulated kinase 1/2 on the suppression of myostatin gene expression induced by basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    Huazhoag Liu; Xiaorong An; Yongfu Chen; Jieping Zhong

    2008-01-01

    Basic fibmblast growth factor (bFGF, FG F-2 ) has an inhibitory effect on the expression of the myostatin gene in murine C2C12 myoblasts, as shown in our recent investigation. To further verify the regulatory effects of bFGF on the myostalin gene and to better understand its mechanism in skeletal muscle, and to promote clinical applications of bFGF to treat skeletal muscle diseases correlated to muscular dystrophy or AIDS and so on, recombinant human bFGF (rh-bFGF) was added into media and stimulated murine C2C12 myoblasts to investigate the dose-dependent effect ofbFGF on suppression of myostatin gene expression and the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulatory mechanism. Simultaneously, complete coding sequence of ovine 18 kDa-bFGF gene was inserted into eukaryotic vector pCMV-neo (originated from pEGFP-N1 vector, from which the EGFP gene has been removed), the recombinant plasmid pCMV-neo-bFGF was harvested and injected into the mouse skeletal muscle of posterior limb. Expression levels of bFGF,myostatin, and ERKI/2 genes in murine C2C12 myoblasts and the skeletal muscle were analyzed by real-time reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. The results showed that bFGFimpaired the expression ofmyostatin gene in a dose-dependent manner in C2C12 cells, with increasing concentration of rh-bFGF,myostatin mRNA declined gradually. In addition, results in skeletal muscle indicated that bFGF also suppressed the expression of the myostatin gene in vivo. Furthermore, we found ERKI/2 participated in the regulatory mechanism of bFGF on the expression of the myostatin gene.

  12. A simple, highly efficient method for heterologous expression in mammalian primary neurons using cationic lipid-mediated mRNA transfection

    Directory of Open Access Journals (Sweden)

    Damian J Williams

    2010-11-01

    Full Text Available Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The postmitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro-transcribed mRNA and the cationic lipid transfection reagent Lipofectamine 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R, a G protein inwardly-rectifying K+ channel (GIRK4 and a dominant-negative G protein α-subunit mutant (GoA G203T indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

  13. Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza.

    Science.gov (United States)

    Garcia, Kevin; Haider, Muhammad Zulqurnain; Delteil, Amandine; Corratgé-Faillie, Claire; Conéjero, Geneviève; Tatry, Marie-Violaine; Becquer, Adeline; Amenc, Laurie; Sentenac, Hervé; Plassard, Claude; Zimmermann, Sabine

    2013-01-01

    Mycorrhizal exchange of nutrients between fungi and host plants involves a specialization and polarization of the fungal plasma membrane adapted for the uptake from the soil and for secretion of nutrient ions towards root cells. In addition to the current progress in identification of membrane transport systems of both symbiotic partners, data concerning the transcriptional and translational regulation of these proteins are needed to elucidate their role for symbiotic functions. To answer whether the formerly described Pi-dependent expression of the phosphate transporter HcPT1.1 from Hebeloma cylindrosporum is the result of its promoter activity, we introduced promoter-EGFP fusion constructs in the fungus by Agrotransformation. Indeed, HcPT1.1 expression in pure fungal cultures quantified and visualized by EGFP under control of the HcPT1.1 promoter was dependent on external Pi concentrations, low Pi stimulating the expression. Furthermore, to study expression and localization of the phosphate transporter HcPT1.1 in symbiotic conditions, presence of transcripts and proteins was analyzed by the in situ hybridization technique as well as by immunostaining of proteins. In ectomycorrhiza, expression of the phosphate transporter was clearly enhanced by Pi-shortage indicating its role in Pi nutrition in the symbiotic association. Transcripts were detected in external hyphae and in the hyphal mantle, proteins in addition also within the Hartig net. Exploiting the transformable fungus H. cylindrosporum, Pi-dependent expression of the fungal transporter HcPT1.1 as result from its promoter activity as well as transcript and protein localization in ectomycorrhizal symbiosis are shown.

  14. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

    Science.gov (United States)

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4+CD25+ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4+ or CD8+ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  15. Construction of lentiviral vectors coexpressing EGFP and rat GDNF gene and transfection rat bone mesenchymal stem cells%EGFP和大鼠GDNF基因共表达的慢病毒载体构建及转染大鼠骨髓间充质干细胞

    Institute of Scientific and Technical Information of China (English)

    张阳; 张志坚; 陈东平; 吴秀丽

    2009-01-01

    为了构建携带增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和胶质细胞源性神经营养因子(glial cell linederived neurotrophic factor,GDNF)基因的慢病毒载体,观察GDNF基因大鼠骨髓间充质干细胞(ral mesenchymal stem cells,rMSC)的表达,本研究采用RT-PCR(reverse transcription-PCR)方法从P0大鼠小脑组织中扩增出GDNF基因编码区636 bp的片段,通过限制性内切酶酶切、T4DNA连接酶连接,将GDNF插入慢病毒转移载体PNL-IRES2-EGFP,构建PNL-GDNF-IRES2-EGFP.在脂质体介导下将构建成功的慢病毒三质粒系统共转染人胚肾细胞系(293T)包装生产慢病毒,测定病毒滴度.感染rMSCs,荧光显微镜下观察EGFP的表达、转导效率,RT-PCR、Western blot方法分别检测GDNF mRNA和蛋白的表达情况.结果显示:GDNF的基因序列经测序后与GeneBank报道的序列完全一致,重组慢病毒载体质粒PNL-GDNF-IRES2-EGFP经双酶切鉴定正确;三质粒共转染293T细胞后荧光激发可见大量绿色荧光,收集、浓缩病毒后测定其滴度为5.3×107pfu/ml;感染rMSCs结果显示:GDNFrMSCs组5 d转导效率为93.3%±3.17%,传代培养4周,下降到81.1%±3.59%,差异具有统计学意义(P<0.01).RT-PCR(real time-PCR)、Western blot显示GDNF成功在rMSCs中表达.本结果表明,我们已经成功构建带有EGFP、GDNF基因的慢病毒载体,并获得GDNF-rMSCs基因工程细胞,但提高该工程细胞外源基因的稳定表达技术仍需进一步探讨.

  16. Construction and migration of rat bone marrow mesenchymal stem cells coexpressing EGFP and CXCR4%共表达EGFP和CXCR4的大鼠骨髓间充质干细胞的构建及其迁移能力

    Institute of Scientific and Technical Information of China (English)

    俞晓岚; 张志坚; 吴秀丽; 黄志新

    2010-01-01

    目的 构建携带增强绿色荧光蛋白(EGFP)及CXCR4慢病毒载体,并实现其在大鼠骨髓间充质干细胞(rMSCs)中的表达,观察转CXCR4基因后对rMSCs迁移能力的影响. 方法 RT-PCR扩增大鼠CXCR4编码区片段,将其插入慢病毒载体质粒PNL-IRES2-EGFP,获得的PNL-CXCR4-IRES2-EGFP与包装及包膜质粒用脂质体法共转染293T细胞,包装生产慢病毒.所获慢病毒转染rMSCs后,用RT-PCR、Western blot、细胞免疫荧光组织化学和流式细胞术检测转CXCR4基因rMSCs组、转空载体rMSCs组中CXCR4表达情况.利用Transwell方法检测两组rMSCs在SDF-1作用下的迁移能力. 结果 双酶切和测序证实,PNL-CXCR4-IRES2-EGFP构建正确,转CXCR4基因rMSCs组CXCR4表达明显增多,在SDF-1α作用下迁移能力明显增强. 结论 成功构建带有EGFP和大鼠CXCR4的慢病毒载体,并获得CXCR4-rMSCs基因工程细胞,为深入研究SDF/CXCR4轴在rMSCs向损伤组织定向迁移中的作用奠定了基础.

  17. Neural crest and mesoderm lineage-dependent gene expression in orofacial development.

    Science.gov (United States)

    Bhattacherjee, Vasker; Mukhopadhyay, Partha; Singh, Saurabh; Johnson, Charles; Philipose, John T; Warner, Courtney P; Greene, Robert M; Pisano, M Michele

    2007-06-01

    The present study utilizes a combination of genetic labeling/selective isolation of pluripotent embryonic progenitor cells, and oligonucleotide-based microarray technology, to delineate and compare the "molecular fingerprint" of two mesenchymal cell populations from distinct lineages in the developing embryonic orofacial region. The first branchial arches-bi-lateral tissue primordia that flank the primitive oral cavity-are populated by pluripotent mesenchymal cells from two different lineages: neural crest (neuroectoderm)- and mesoderm-derived mesenchymal cells. These cells give rise to all of the connective tissue elements (bone, cartilage, smooth and skeletal muscle, dentin) of the orofacial region (maxillary and mandibular portion), as well as neurons and glia associated with the cranial ganglia, among other tissues. In the present study, neural crest- and mesoderm-derived mesenchymal cells were selectively isolated from the first branchial arch of gestational day 9.5 mouse embryos using laser capture microdissection (LCM). The two different embryonic cell lineages were distinguished through utilization of a novel two component transgenic mouse model (Wnt1Cre/ZEG) in which the neural crest cells and their derivatives are indelibly marked (i.e., expressing enhanced green fluorescent protein, EGFP) throughout the pre- and post-natal lifespan of the organism. EGFP-labeled neural crest-derived, and non-fluorescent mesoderm-derived mesenchymal cells from the first branchial arch were visualized in frozen tissue sections from gestational day 9.5 mouse embryos and independently isolated by LCM under epifluorescence optics. RNA was extracted from the two populations of LCM-procured cells, and amplified by double-stranded cDNA synthesis and in vitro transcription. Gene expression profiles of the two progenitor cell populations were generated via hybridization of the cell-type specific cRNA samples to oligo-based GeneChip microarrays. Comparison of gene expression

  18. 过表达巨噬细胞移动抑制因子对子宫颈癌SiHa细胞中白细胞介素8及基质金属蛋白酶9表达的影响%Effects of over-expression of macrophage migration inhibitory factor on the expression of interleukin-8 and martix metalloproteinase-9 of human cervical cancer SiHa cells

    Institute of Scientific and Technical Information of China (English)

    郭红霞; 吴素慧; 贾睿; 尚海霞

    2013-01-01

    Objective To investigate the effects of macrophage migration inhibitory factor (MIF) overexpression on the expression of interleukin-8 (IL-8),martix metalloproteinase-9 (MMP-9) and invasion of human cervical cancer SiHa cells.Methods Chemical synthesis MIF eDNA gene,designed primer sequence including XhoI and BamHI enzyme sites,MIF gene was amplified by polymerase chain reaction (PCR),constructed eukaryotic expression vector pEGFP-N1/MIF and transfected into SiHa cells using Lipofectamine and won over-expression of MIF.The expression of MIF in supernatant fluid was detected by ELISA,the expression of MIF,IL-8,MMP-9 in both mRNA and protein levels were detected by real-time fluorescence quantitative-PCR and immunocytochemistry respectively.The effect of over-expressed MIF on migration was detected by Boyden small chamber.Results The expression of protein in supernatant fluid transfected with pEGFP-N1/MIF was significantly increased (Fgroup =8267.564,P < 0.01),the expression of MIF,IL-8,MMP-9 in both mRNA and protein in SiHa cells transfected with pEGFP-N1/MIF were significantly increased (F values were 7019.619,2148.094,3303.540,1565.114,2807.300,523.466,P < 0.01),and there was a positive correlation among MIF,IL-8,MMP-9 expression in both mRNA and protein (r values were 0.865,0.895,0.934,0.908,P < 0.01).Invasion ability in SiHa cells transfected with pEGFP-N1/MIF was obviously increased (F=3430.898,P< 0.01).Conclusion The over-expression MIF gene in SiHa cells can promote cervical cancer cell invasion and metastasis of ability,which could be associated with the upregulation of IL-8 and MMP-9 expression.%目的 研究过表达巨噬细胞移动抑制因子(MIF)对子宫颈癌SiHa细胞中白细胞介素8(IL-8)、基质金属蛋白酶9(MMP-9)表达及细胞侵袭迁移能力的影响.方法 化学合成MIF cDNA,设计含Xhol和BamHI酶切位点的引物序列,利用聚合酶链反应(PCR)方法 扩增MIF基因片段,构建人pEGFP-N1/MIF真核表达载体,

  19. Construction of eukaryotic vector of monkey B virus glycoprotein D gene and the gD gene expression%猕猴B病毒囊膜蛋白gD基因真核细胞表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    王新; 易思萌; 刘慧芳; 马凯; 范君文; 马雨楠; 游颖; 孙兆增

    2015-01-01

    Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.%目的:构建猕猴B病毒囊膜蛋白gD的真核表达载体,并且检测其在293T细胞内的表达情况。方法首先通过基因合成手段获得含B病毒gD蛋白的基因片段,在经由PstⅠ和NotⅠ双酶切后连接到pEGFP-N3载体,随后将构建的pEGFP-N3-GD重组质粒转染到人胚胎肾上皮细胞系293T细胞。再用Western blot检测所提蛋白其在细胞内的表达情况,并用激光共聚焦分析其在细胞内的表达定位情况。结果成功获得携带gD基因的阳性重组质粒pEGFP-N3-GD,且pEGFP-N3-GD重组质粒能在293T细胞的表面正常表达。结论利用真核表达系统,既能够在细胞表面产生B病毒gD蛋白的特异性重组抗原,而且可用于B检测抗原的制备。

  20. Rat adipose-derived stromal cells expressing BMP4 induce ectopic bone formation in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Lin LIN; Xin FU; Xin ZHANG; Lian-xu CHEN; Ji-ying ZHANG; Chang-long YU; Kang-tao MA; Chun-yan ZHOU

    2006-01-01

    Aim: Bone morphogenetic protein 4 (BMP4) is one of the main local contributing factors in callus formation in the early phase of fracture healing. Adipose-derived stromal cells (ADSC) are multipotent cells. The present study was conducted to investigate the osteogenic potential of ADSC when exposed to adenovirus containing BMP4 cDNA (Ad-BMP4). Methods: ADSC were harvested from Sprague-Dawley rats. After exposure to Ad-BMP4, ADSC were assessed by alkaline phos-phatase activity (ALP) assay, RT-PCR and von Kossa staining. BMP4 expression was assessed by RT-PCR, immunofluorescence and Western blot analysis. ADSC transduced with Ad-BMP4 were directly injected into the hind limb muscles of athymic mice. ADSC Ad-EGFP(enhanced green fluorescence protein) served as controls. All animals were examined by X-ray film and histological analysis. Results: The expression of BMP4 was confirmed at both mRNA and protein levels. The expression of the osteoblastic gene, ALP activity and von Kossa staining confirmed that ADSC transduced with Ad-BMP4 underwent rapid and marked osteoblast differentiation, whereas ADSC transduced with Ad-EGFP and cells left alone displayed no osteogenic differentiation. X-ray and histological examination confirmed new bone formation in athymic mice transplanted with ADSC transduced with Ad-BMP4. Conclusion: Our data demonstrated successful osteogenic differentiation of ADSC transduced with Ad-BMP4 in vitro and in vivo. ADSC may be an ideal source of mesenchyme lineage stem cells for gene therapy and tissue engineering.

  1. Expression Silence of DNA Repair Gene hMGMT Induced by RNA Interference

    Institute of Scientific and Technical Information of China (English)

    LI Xiu-ying; LAI Yan-dong

    2007-01-01

    Objective: MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods: The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUC19 to get pU6-MGMTi, co-transfected with pEGFP-C1 into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results: hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion: MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.

  2. Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation

    Directory of Open Access Journals (Sweden)

    Moen Ingrid

    2012-01-01

    Full Text Available Abstract Background The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. Methods 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7, Group 2 to 7 daily HBO treatments (both 2.5bar, 100% O2, à 90 min, whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. Results The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. Conclusions The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth

  3. Construction of Eukaryotic Expression Vector and Sequence Analysis of Antimicrobial Peptide Gene Shiva 1a%抗菌肽Shiva1a基因真核表达载体的构建及序列分析

    Institute of Scientific and Technical Information of China (English)

    宫晓炜; 郑福英; 蔺国珍; 曹小安; 王光华; 周继章; 才学鹏

    2011-01-01

    为了探讨天蚕类抗菌肽在动物早期抗感染过程中的作用机理,本研究以Shiva 1a基因的成熟肽为模板设计4条引物,利用重叠延伸PCR技术获得目的基因,并在C端添加6×His的标签.将此序列与真核表达载体pIRES2 -EGFP进行重组,构建pIRES2-EGFP-Shiva 1a重组表达质粒,对重组质粒进行酶切和测序鉴定后,采用阳离子脂质体转染将重组质粒转染到CHO-K1细胞,荧光显微镜观察其表达情况.通过生物信息学软件对抗菌肽Shiva 1a的二级结构和三级结构进行预测分析.其结果为进一步研究Shiva 1a的抗菌活性和在动物抗病育种方面的应用奠定了基础.%To explore the effect mechanism of cecropin-class lytic peptide at early stage of infection. The mat peptide of Shiva la was amplified by overlap extension PCR and the C-terminus contained 6×His-marker. The gene sequence were recombi-nant with eukaryotic expression vector pIRES2-EGFP. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmid pIRES2-EGFP-Shiva la was transfected into CHO-K1 cells by liposomes. The expression of the the recombinant plasmid pIRES2-EGFP-Shiva 1a was observed by fluorescence microscope. At the same time, the secondary structure and 3D structure were predicted by bioinformatics tools. The results lay the foundation in research of antimicrobial activities and applications of the antimicrobial peptide Shiva la in breeding for disease resistance of animals.

  4. A rapid and efficient method to express target genes in mammalian cells by baculovirus

    Institute of Scientific and Technical Information of China (English)

    Tong Cheng; Chen-Yu Xu; Ying-Bin Wang; Min Chen; Ting Wu; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: To investigate the modification of baculovirus vector and the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of Spodoptera frugiperta (Sf9) cells infected by recombinant baculoviruses.METHODS: Two recombinant baculoviruses (BacV-CMVEGFPA, BacV-CMV-EGFPB) containing CMV-EGFP expression cassette were constructed. HepG2 cells were directly incubated with the culture supernatant of Sf9 cells infected by recombinant baculoviruses, and reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM). The optimal transduction conditions were investigated by FCM assay in HepG2 cells. Gene-transfer and expression efficiencies in HepG2 or CV1 cells by baculovirus vectors were compared with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Twenty different mammalian cell lines were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the culture supernatant of infected Sf9 cells.RESULTS: CMV promoter could directly express reporter genes in Sf9 cells with a relatively low efficiency. Target cells incubated with the 1:1 diluted culture supernatant (moi=50) for 12 h at 37 ℃ could achieve the highest transduction and expression efficiencies with least impairment to cell viability. Under similar conditions the baculovirus vector could achieve the highest gene-transfer and expression efficiency than lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Most mammalian cell lines could be transduced with recombinant baculovirus. In primate adherent culture cells the recombinant baculovirus could arrive the highest infection and expression efficiencies, but it was not very satisfactory in the cell lines from mice and suspended culture cells.CONCLUSION: Mammalian cells incubated with the culture supernatant of infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign

  5. Effect of overexpression of wild-type p53 on POLD1 expression and malignant cell behavior in human hepatocellular carcinoma cell line SMMC-7721%野生型p53对肝癌细胞POLD1基因表达及细胞恶性表型的影响

    Institute of Scientific and Technical Information of China (English)

    韦长元; 刘起理; 廖柳凤; 徐恒; 谭晓虹

    2011-01-01

    AIM: To investigate the impact of overexpres-sion of wild-type p53 on cell proliferation and malignant phenotype in human hepatocellular carcinoma cell line SMMC-7721 and to explore possible mechanism involved.METHODS: Enhanced green fluorescence protein gene-containing eukaryotic expressionplasmids expressing p53-specific small interfering RNA (shRNA) (p53-siRNA) or wild-type p53 (pEGFP-p53) were constructed and introduced into SMMC-7721 cells by Lipofection-2000-mediated transfection. Meanwhile, the pEGFP-Cl empty vector was also transfected into SMMC-7721 cells. Cell lines stably expressing p53-siRNA, pEGFP-p53 or pEGFP-Cl were screened in medium containing G418. After transfection, the expression of p53 and POLD1 mRNAs was detected by RT-PCR. The changes in malignant cell behavior were determined by cell growth curve determination and colony formation assay.RESULTS: Compared to control SMMC-7721 cells, p53 mRNA expression was increased and POLD1 gene expression was decreased in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene, while p53 mRNA expression was reduced and POLD1 mRNA expression was increased in SMMC-7721 cells transfected with the plasmid carrying p53-siRNA. MTT results showed that cell growth rate was faster in SMMC-7721 cells transfected with the plasmid carrying p53-siRNA than in control SMMC-7721 cells, but was slower in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene than in control cells. Colony formation assay showed that colony formation rate was lower in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene than in control cells (38.1% vs 52.6%, P < 0.05), but was higher in cells tranfected with the plasmid carrying p53-siRNA than in control cells (72.6% vs 52.6%, P < 0.05). High expression of wild-type p53 inhibited POLD1 transcription and cell proliferation, while low expression of wild-type p53 promoted POLD1 transcription and cell proliferation.CONCLUSION: Wild-type p53

  6. Natural Loss of eyeless/Pax6 Expression in Eyes of Bicyclus anynana Adult Butterflies Likely Leads to Exponential Decrease of Eye Fluorescence in Transgenics

    Science.gov (United States)

    Monteiro, Antónia

    2015-01-01

    Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals. PMID:26173066

  7. Natural Loss of eyeless/Pax6 Expression in Eyes of Bicyclus anynana Adult Butterflies Likely Leads to Exponential Decrease of Eye Fluorescence in Transgenics.

    Science.gov (United States)

    Das Gupta, Mainak; Chan, Sam Kok Sim; Monteiro, Antónia

    2015-01-01

    Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals.

  8. Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies

    Science.gov (United States)

    Simpson, Sean; Collins, Bruce; Sommer, Jeff; Petters, Robert M.; Caballero, Ignacio; Platt, Jeff L.

    2017-01-01

    Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3–5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model. PMID:28081156

  9. Cloning of the Bovine Natural Resistance-associated Macrophage Protein Ⅰ Gene Isoform (NRA MPI-ISO) and Its Expression in the Cells and the Tissues%牛巨噬细胞天然抗性蛋白Ⅰ基因可变剪接体(NRAMPI-ISO)的克隆及在细胞和组织中的表达

    Institute of Scientific and Technical Information of China (English)

    程祥; 邓捷; 孟书燕; 来威锋; 王华岩

    2011-01-01

    巨噬细胞天然抗性蛋白1 (NRAMP1)可抑制结核分枝杆菌(Mycobacterium)和布氏杆菌(Brucella)等多种胞内寄生病原菌的感染,提高动物机体的抗病能力.本研究从秦川牛脾脏中克隆了NRA MP1基因的一种可变剪接体NRA MPI-ISO,序列分析表明,NRA MPI-ISO比NRAMP1多了第3个内含子,从而导致编码序列提前终止于第3个内含子,NRA MPI-ISO编码200个氨基酸.为了探索可变剪接体NRAMPI-ISO的表达情况,本研究分别构建了原核表达载体pET-41-NRA MPI-ISO和真核表达载体pEGFP-NRA MPI-ISO.原核表达载体pET-41 -NRA MPI-ISO可在不同浓度的IPTG诱导下在大肠杆菌BL21中高效表达.真核表达载体pEGFP-NRA MPI-ISO转染牛成纤维细胞后,EGFP-NRAMP 1-ISO融合蛋白分布在细胞核和细胞质中,而正常的NRAMP1蛋白只分布在细胞质的溶酶体膜周围.半定量RT-PCR检测表明,NRA MPI-ISO基因在心、脾脏、肺脏等组织有较高的表达,而在脑、胰、生殖嵴中的表达量相对较低.本研究为进一步研究NRA MPI-ISO基因生物学功能提供了重要信息.%Natural resistance-associated macrophage protein 1 (NRAMP1) can defense the invasion of the intracellular bacteria such as Mycobacterium and Brucella to improve the ability of resisting disease, hi this study we cloned a NRAMP1 isoform from the spleen of Qinchuan Bos Taurus. Sequence analysis showed that NRAMP1 isoform had a third intron in comparison with NRAMP1, which resulted in the protein only containing 200 amino acids because the translation stopped in the third intron. In order to get further study on the expression of NRAMP1-ISO, we constructed the prokaryotic expression vector pET-41-NRAMP1-ISO and the eukaryotic expression vector pEGFP-NRAMPl-ISO. The prokaryotic expression vector pET-41-NRAMPl-ISO could efficiently express in the BL21 Escherichia coli under different concentrations of the IPTG. The eukaryotic expression vector pEGFP-MRAMP1-ISO could both express in the

  10. A maize-specifically expressed gene cluster in Ustilago maydis.

    Science.gov (United States)

    Basse, Christoph W; Kolb, Sebastian; Kahmann, Regine

    2002-01-01

    The corn pathogen Ustilago maydis requires its host plant maize for development and completion of its sexual cycle. We have identified the fungal mig2-1 gene as being specifically expressed during this biotrophic stage. Intriguingly, mig2-1 is part of a gene cluster comprising five highly homologous and similarly regulated genes designated mig2-1 to mig2-5. Deletion analysis of the mig2-1 promoter provides evidence for negative and positive regulation. The predicted polypeptides of all five genes lack significant homologies to known genes but have characteristic N-terminal secretion sequences. The secretion signals of mig2-1 and mig2-5 were shown to be functional, and secretion of a full length Mig2-1-eGFP fusion protein to the extracellular space was demonstrated. The central domains of the Mig2 proteins are highly variable whereas the C-termini are strongly conserved and share a characteristic pattern of eight cysteine residues. The mig2 gene cluster was conserved in a wide collection of U. maydis strains. Interestingly, some U. maydis isolates from South America had lost the mig2-4 gene as a result of a homologous recombination event. Furthermore, the related Ustilago scitaminea strain, which is pathogenic on sugar cane, appears to lack the mig2 cluster. We describe a model of how the mig2 cluster might have evolved and discuss its possible role in governing host interaction.

  11. Using inositol as a biocompatible ligand for efficient transgene expression.

    Science.gov (United States)

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

  12. Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors.

    Directory of Open Access Journals (Sweden)

    Tihana Jovanic

    Full Text Available Somatic hypermutation (SHM of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C to deoxyuridine (U, catalysed by the activation induced deaminase (AID. Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue, followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.

  13. Synthesis, depletion and cell-type expression of a protein from the male accessory glands of the dengue vector mosquito Aedes aegypti.

    Science.gov (United States)

    Alfonso-Parra, Catalina; Avila, Frank W; Deewatthanawong, Prasit; Sirot, Laura K; Wolfner, Mariana F; Harrington, Laura C

    2014-11-01

    Aedes aegypti males transfer sperm and seminal fluid proteins (Sfps), primarily produced by male accessory glands (AGs), to females during mating. When collectively injected or transplanted into females, AG tissues and/or seminal fluid homogenates have profound effects on Aedes female physiology and behavior. To identify targets and design new strategies for vector control, it is important to understand the biology of the AGs. Thus, we examined characteristics of AG secretion and development in A. aegypti, using the AG-specific seminal fluid protein, AAEL010824, as a marker. We showed that AAEL010824 is first detectable by 12h post-eclosion, and increases in amount over the first 3 days of adult life. We then showed that the amount of AAEL0010824 in the AG decreases after mating, with each successive mating depleting it further; by 5 successive matings with no time for recovery, its levels are very low. AAEL010824 levels in a depleted male are replenished by 48 h post-mating. In addition to examining the level of AAEL010824 protein, we also characterized the expression of its gene. We did this by making a transgenic mosquito line that carries an Enhanced Green Fluorescence Protein (EGFP) fused to the AAEL0010824 promoter that we defined here. We showed that AAEL010824 is expressed in the anterior cells of the accessory glands, and that its RNA levels also respond to mating. In addition to further characterizing AAEL010824 expression, our results with the EGFP fusion provide a promoter for driving AG expression. By providing this information on the biology of an important male reproductive tissue and the production of one of its seminal proteins, our results lay the foundation for future work aimed at identifying novel targets for mosquito population control.

  14. Attenuated Salmonella typhimurium as a carrier for prokaryotic and eukaryotic expression vectors%减毒沙门氏菌作为原核表达宿主菌和真核表达载体的研究

    Institute of Scientific and Technical Information of China (English)

    迪卡; 陈雪燕; 帅江冰; 陈宁; 方维焕

    2006-01-01

    An attenuated Salmonella enterica serovar typhimurium strain as the host for recombinant prokaryotic and eukaryotic vectors containing the enhanced green fluorescent protein (eGFP) gene was examined by fluorimetric and fluorescent microscopic methods. Expression of eGFP was dependent on the concentration of IPTG used for induction, being optimal at 0.5 mmol. L-1 . Further increase to 0.75mmol. L-1 did not increase the fluorescence output. This dose-dependent induction was not apparent when E. coli was used as the host strain. The expression was higher in S. typhimurium than in E. coli typhimurium strain was invasive, though less than its parent strain, into the HeLa cells and able to deliver the recombinant eukaryotic plasmid pcDNA3-eGFP for expression of eGFP as shown by fluorescing cells 48 h after transfection. The results of this experiment also demonstrate the utility of direct measurement of fluorescence and optical density in a multifunctional microplate-based spectrophotometric reader, allowing high throughput multiple quantitative comparisons of eGFP expression by different host strains or the same strain under different conditions or even different expression vectors.%本研究以增强型绿色荧光蛋白(eGFP)为报告基因,探讨了减毒沙门氏菌作为原核表达宿主菌和真核表达载体的可行性.通过多功能分光光度仪测定细菌的OD600和eGFP在大肠杆菌和减毒沙门氏菌中的表达量,结果表明:eGFP在沙门氏菌中的表达量依赖于IPTG的浓度,最佳浓度为0.5 mmol·L-1,增加浓度至0.75 mmol·L-1不能增加eGFP的表达量;而该现象在大肠杆菌中不明显.当IPTG浓度等于或大于0.5 mmol·L-1时,沙门氏菌中eGFP的表达量显著高于大肠杆菌.若以相对荧光度(1个OD600单位的荧光值,即eGFP相对表达量)表示,两种宿主菌的最佳诱导时间均为3 h.Hela细胞侵袭力试验表明该减毒沙门氏菌仍具有侵袭力,同时以脂质体转染为对

  15. Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.

  16. Ascl3 expression marks a progenitor population of both acinar and ductal cells in mouse salivary glands.

    Science.gov (United States)

    Bullard, Tara; Koek, Laurie; Roztocil, Elisa; Kingsley, Paul D; Mirels, Lily; Ovitt, Catherine E

    2008-08-01

    Ascl3, also know as Sgn1, is a member of the mammalian achaete scute (Mash) gene family of transcription factors, which have been implicated in cell fate specification and differentiation. In the mouse salivary gland, expression of Ascl3 is restricted to a subset of duct cells. Salivary gland function depends on the secretory acinar cells, which are responsible for saliva formation, and duct cells, which modify the saliva and conduct it to the oral cavity. The salivary gland ducts are also the putative site of progenitor cells in the adult gland. Using a Cre recombinase-mediated reporter system, we followed the fate of Ascl3-expressing cells after the introduction of an EGFP-Cre expression cassette into the Ascl3 locus by homologous recombination. Lineage tracing shows that these cells are progenitors of both acinar and ductal cell types in all three major salivary glands. In the differentiated progeny, expression of Ascl3 is down-regulated. These data directly demonstrate a progenitor-progeny relationship between duct cells and the acinar cell compartment, and identify a population of multipotent progenitor cells, marked by expression of Ascl3, which is capable of generating both gland cell types. We conclude that Ascl3-expressing cells contribute to the maintenance of the adult salivary glands.

  17. 分枝杆菌膜锚定表达载体的构建与亚细胞定位分析%The construction and sub-cellular localization analysis of novel mycobacterial membrane-anchored expression vector

    Institute of Scientific and Technical Information of China (English)

    王鑫; 范小勇; 马辉; 曲勍; 朱越雄

    2011-01-01

    Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis(Mtb) 19×103 lipoprotein (19SS) was synthesized and was then cloned into the downstream of pfurAma mutant to generate the mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein(EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce recombinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western blot and flow cytometry sorting(FCS), and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The novel mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×103 lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could also be used to research cytophagy in cell model and mycobacterial colony and translocation in animal immunization as model indicator

  18. Effect of SMYD3 over-expression on DNMT3B levels and proliferation ability in human cholangiocarcinoma cell line FRH0201%上调SMYD3对人胆管癌FRH0201细胞中DNMT3B表达及细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    程帝; 李志花; 陈汝福; 郭宁; 廖巧芳; 郑礼平; 周泉波; 周嘉嘉

    2012-01-01

    目的:研究人胆管癌细胞株FRH0201中SET和MYND结构域含有蛋白3(SET and MYND domain-containing protein 3,SMYD3)过度表达对DNA甲基化转移酶3B(DNA methyltransferase 3B,DNMT3B)表达及细胞增殖能力的影响.方法:瞬时转染SMYD3真核表达质粒后,RT-PCR检测细胞中DNMT3B mRNA水平的变化;Western blotting检测细胞中DNMT3B蛋白水平的变化;CCK-8检测细胞增殖能力的改变;流式细胞术检测细胞周期的改变.结果:以未处理组为对照,胆管癌细胞FRH0201在转染pEGFP-C3-SMYD3质粒后,DNMT3B蛋白及mRNA表达均显著上升(P<0.01);细胞的增殖能力显著提高、细胞增殖速度加快(P<0.05);进入G2/M期的细胞明显增多(P<0.05).结论:过度表达SMYD3,可引起细胞中DNMT3B的表达上调并增强细胞增殖能力.%AIM:To explore ihe effect of SET and MYND domain - containing protein 3 ( SMYD3 ) over - expression on the expression of DNA methyltransferase 3B (DNMT3B) and the proliferation ability in human cholangiocarci-noma cell line FRH0201. METHODS; Transient transfection of SMYD3 eukaryotic expression plasmid pEGFP - C3 - SMYD3 into human cholangiocarcinoma cell line FRH0201 was performed. The expression of DNMT3B at mRNA and protein levels was detected by RT - PCR and Western blotting,respectively. Cell proliferation was examined by CCK -8 method and cell cycle situation was checked by flow cytometry. RESULTS; After transfected with SMYD3 eukaryotic expression plasmid pEGFP - C3 - SMYD3 , the over - expression of SMYD3 in FRH0201 cells was observed. Compared with the un-transfected cells, the expression of DNMT3B was significantly increased (P <0. 01) , the proliferation rate was obviously accelerated (P<0. 05) and the number of the cells in G2/M phase was significantly increased (P<0. 05) in FRH0201 cells transiently transfected with pEGFP - C3 - SMYD3 plasmid. CONCLUSION; The transient transfection of pEGFP -C3 - SMYD3 plasmid induces over - expression of DNMT3B and

  19. Construction of recombinant lentivirus expression vector carrying MagA and its in vino expression%磁共振报告基因magA的慢病毒载体质粒构建及体外表达

    Institute of Scientific and Technical Information of China (English)

    秦勇; 蔡金华; 郑鹤琳; 刘官信; 王世一; 刘波

    2011-01-01

    目的 构建携带磁共振报告基因magA的慢病毒载体质粒,初步检测其体外转铁效应.方法 采用人工DNA合成技术合成目的 基因magA,DNA重组技术将magA基因连接入慢病毒表达载体质粒pLenti-EGFP,酶切及DNA测序鉴定重组质粒pLenti-EGFP/magA的准确性.包装、包膜及重组质粒共转染293FT细胞包装慢病毒,收集包被magA基因的慢病毒上清并感染293T靶细胞,细胞培养基内加入500 μmol/L枸橼酸铁连续4次传代,荧光显微镜观察并提取细胞行台盼蓝排除实验和普鲁士蓝染色,同时设立对照组行同样方法实验.结果酶切和DNA测序分析证实magA基因准确克隆入慢病毒表达载体设计位点,合成目的 基因序列与GenBank中magA序列完全一致.荧光显微镜下观察包装细胞293FT细胞质内有大量绿色荧光表达,病毒滴度达到108 Tu/μl.慢病毒感染293T靶细胞后,细胞内稳定表达EGFP,且效率>80%.实验组及对照组台盼蓝拒染率分别为(92.80±2.65)、(93.50±1.29),2组拒染率差异无统计学意义(P>0 05).普鲁士蓝染色发现实验组细胞内有大量蓝染铁颗粒形成,对照组呈阴性.结论 成功构建了携带磁共振报告基因magA的慢病毒表达载体,证实了magA基因在哺乳动物293T靶细胞内的转铁作用.%Objective To construct a recombinant lentivirus expression vector encoding magA, a new MRI reporter gene, and to determine the iron-transporting effect of in vitro expressed magA. Methods The gene sequence of magA was synthesized by synthetic DNA technology, and then inserted into the lentivirus expression vector pLenti-EGFP by recombinant DNA technique. The recombinant lentivirus expression vector pLenti-EGFP/magA was verified by enzyme digestion and DNA sequencing. Then the recombinant lentivirus was packaged in the 293FT cells by co-transfecting with packaging plasmid, envelope plasmid, and lentiviral vector plasmid. The 293T cells were transfected with the packaged

  20. The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

    Institute of Scientific and Technical Information of China (English)

    Yi-qin WANG; Yu-cheng YANG; Wen-lu ZHANG; Su-ling HONG

    2007-01-01

    To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.

  1. NF-kB activation and its downstream target genes expression after heavy ions exposure

    Science.gov (United States)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  2. Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum

    Directory of Open Access Journals (Sweden)

    Vahid NASIRI

    2016-10-01

    Full Text Available Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge.Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected.Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development. 

  3. PiggyBac转座酶转基因斑马鱼模型的初步建立%Establishment of a PiggyBac transposase -expressing transgenic Danio rerio model

    Institute of Scientific and Technical Information of China (English)

    曹守莹; 白长存

    2016-01-01

    Objective To establish a PiggyBac Transposase -expressing transgenic Danio rerio model for studying genetic modi-fication mediated by tansposon in zebrafish .Methods PiggyBac Transposase gene and the Enhanced green fluorescent protein ( EG-FP) gene were driven by CMV promoter and transgenic zebrafish were created by microinjection .Results PiggyBac transposase -ex-pressing transgenic zebrafish were obtained by detecting the ubiquitous expression of EGFP in F 0 zebrafish larvae after microinjection . The PiggyBac transposase gene inserted in the genome could be transmitted to F 1 progeny .Regulated gene expression by CMV promoter could be recapitulated in transgenic fish and pCMV -hyPBase-IRES-EGFP transgenic zebrafish was obtained .Conclusion A Pig-gyBac transposase -expressing transgenic zebrafish model was established .This hyperactive piggyBac transposase model expanded the utility of the piggyBac transposon for applications in zebrafish genetics and gene therapy .%目的:构建PiggyBac转座酶转基因斑马鱼模型。方法利用Cytomegalovirus ( CMV)启动子驱动PiggyBac转座酶基因与增强型绿色荧光蛋白( EGFP)基因共表达,通过显微注射后绿色荧光蛋白的表达筛选PiggyBac转座酶转基因斑马鱼。结果建立了能够传代的PiggyBac转座酶转基因斑马鱼模型,通过对增强型绿色荧光蛋白( EGFP)进行定期观察确定了Pig-gyBac转座酶的时间和空间表达方式。提取转基因斑马鱼基因组DNA,PCR鉴定确定转基因斑马鱼基因组DNA中有PiggyBac转座酶基因片段插入。结论初步建立了PiggyBac转座酶转基因斑马鱼模型,为PB转座子在斑马鱼中的应用提供了重要的工具资源。

  4. Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters.

    Science.gov (United States)

    Marchegiani, Elisabetta; Sidhu, Yaadwinder; Haynes, Ken; Lebrun, Marc-Henri

    2015-06-01

    Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a β-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.

  5. Heterogeneity of astrocytes: from development to injury - single cell gene expression.

    Directory of Open Access Journals (Sweden)

    Vendula Rusnakova

    Full Text Available Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+ and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50. The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20 was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50. Within 14 days after ischemia (D3, D7, D14, additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3, transcriptionally active early reactive glia (mainly from D7 and permanent reactive glia (solely from D14. Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

  6. Prokaryotic expression, purification, and production of polyclonal antibody against novel human serum inhibited related protein I (SI1).

    Science.gov (United States)

    Ma, Mingxing; Ma, Jie; Shi, Yinghui; Wu, Hong; Zhao, Wenxiu; Huang, Weiwei; Jiao, Yang; Tan, Deyong

    2010-02-01

    A novel serum inhibited related gene (SI1) has been cloned in our lab by using mRNA differential display analysis of U251 cells in the presence or absence of serum, the expression of SI1 was dramatically inhibited by the addition of serum to serum starved cells. Previous reports suggested the potential significance of SI1 in regulating the cell cycle. In this study, the plasmid construction, protein expression and purification, as well as the generation of anti-SI1 polyclonal antibody are described. A full-length cDNA of Si1 was inserted in a prokaryotic expression plasmid pET28-b(+) and efficiently expressed in E. coli Rosetta (DE3) strain after induction by isopropyl-b-D: -thiogalactoside. The expressed 6His-tagged SI1 fusion protein was purified by Ni(+) affinity column and then used to immunize Balb/C mice, and the anti-SI1 polyclonal antibody was purified by protein A column. To determine the sensitivity and specificity of the antibody against SI1, a cell lysate of pEGFP-N2-SI1 plasmid transiently transfected Hela cell was identified by anti-GFP monoclonal antibody and anti-SI1 polyclonal antibody. Both the GFP-SI1 fusion protein and endogenous SI1 protein in Hela cell can be recognized by the anti-SI1 polyclonal antibody. The anti-SI1 polyclonal antibody will provide a useful tool for further characterization of SI1.

  7. Construction of human BMP2-IRES-HIF1αmu adenovirus expression vector and its expression in mesenchymal stem cells.

    Science.gov (United States)

    Liu, Danping; Hu, Liang; Zhang, Zheng; Li, Quan Ying; Wang, Guoxian

    2013-02-01

    The present study aimed to construct a novel recombinant adenovirus expression vector Ad-BMP2-IRES-HIF1αmu that expresses human bone morphogenetic protein (BMP2) and mutant hypoxia-inducible factor 1α, and investigated its effects in promoting neogenesis of bone and angiogenesis. The recombinant adenovirus BMP2, HIF1αmu and pIRES2-EGFP expression vectors were constructed and transfected into HEK293A cells. The groups were divided into group A, transfection with Ad-BMP2-IRES-HIF1αmu; group B, transfection with Ad-HIF1αmu-IRES-hrGFP-1; group C, transfection with Ad-BMP2-IRES-hrGFP-1; group D, transfection with Ad-IRES-hrGFP-1; group E, not transfected. Adenovirus liquid was transferred into rabbit mesenchymal stem cells (MSCs) pretreated with dexamethasone at the best multiplicity of infection (MOI). The mRNA and protein expression of BMP2 and HIF1α were detected by RT-PCR and western blot analysis. Adenovirus was successfully packaged. The expression level of HIF1α mRNA in group A and B was markedly higher than that in groups C, D and E, showing a significant difference (PBMP2 mRNA between group A and C (PBMP2 in group A and C was markedly higher than that in groups B, D and E (PBMP2-IRES-HIF1αmu adenovirus expression vector was successfully constructed and the experimental groups formed bone and blood vessels prior to the positive and negative control groups.

  8. Expressing functional siRNAs in mammalian cells using convergent transcription

    Directory of Open Access Journals (Sweden)

    Dawes Ian W

    2003-11-01

    Full Text Available Abstract Background The use of small interfering RNAs (siRNAs as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi-mediated gene silencing. Results With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. Conclusion The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences

  9. Construction of enkaryotic expression vector of major histocompatibility complex class Ⅰ -related chain A and establishment of its stable transfected%MHC-Ⅰ类链相关基因A真核表达载体的构建及稳定转染舌鳞癌细胞的实验研究

    Institute of Scientific and Technical Information of China (English)

    李超; 杨丹; 石芳琼; 李跃辉; 陈新群; 翦新春; 蒋灿华

    2011-01-01

    目的 构建人类MHC-Ⅰ类链相关基因A(MICA)的真核表达载体,转染人舌鳞癌脑高转移Tca8113-Tb细胞,建立稳定过表达MICA基因的口腔鳞癌细胞系.方法 采用PCR技术扩增pCMV-SPORT6-MICA中编码MICA基因的cDNA序列,重组至有绿色荧光蛋白标记的真核表达载体pEGFP-N1,构建最终的表达载体pEGFP-N1-MICA,脂质体法转染Tca8113-Tb细胞,G418筛选,荧光显微镜下观察绿色荧光蛋白的表达,有限稀释法建立稳定过表达MICA基因的Tca8113-Tb细胞系,RT-PCR、real time PCR和免疫细胞化学检测MICA在该细胞中的表达.结果 通过PCR技术获取TMICA基因并成功克隆入载体,测序鉴定该序列与GenBank中的序列相同.转染的细胞可见绿色荧光蛋白表达,RT-PCR、real time PCR及免疫细胞化学检测到目的 基因MICA在转染细胞中为过表达.结论 pEGFP-N1-MICA真核表达载体的成功构建与稳定转染Tca8113-Tb细胞系的建立,为进一步研究该基因的功能奠定了良好的实验基础.%Objective To construct the eukaryotic expression vector, encoding major histocompatibility complex class Ⅰ -related chain A gene(MICA), for the further research of transfecting Tca8113-Tb cell line(a metastatic cell line of brain metastasis from human tongue cancer Tea8113 cells in nude mouse), and to establish a stable MICA overexpression oral squamons cell line. Methods eDNA of MICA gene from pCMV-SPORT6-MICA was amplified by PCR,and subcloned into eukaryotic expression vector pEGFP-N1 marked with green fluorescent protein (GFP). The recombinant plasmid was sequenced and transfected into Tca8ll3-Tb cell line by lipofectamineTM 2000. After screen culture by C418, stable tranfected Tca8ll3-Tb cell line was established using definite dilution method. The expressions of GFP protein was viewed directly with fluorescence microscopy and the overexpression of MICA was identified by RT-PCR,real time PCR and immunocytochemistry. Results The MICA gene was

  10. CD147慢病毒表达载体的构建及稳定转染A549细胞系的建立%Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

    Institute of Scientific and Technical Information of China (English)

    杨绍兴; 汤传昊; 王思涵; 宋三泰; 刘晓晴

    2012-01-01

    背景与目的 CD147是一类位于肿瘤细胞膜表面的跨膜糖蛋白,可促进肿瘤的浸润和转移.本研究拟构建CD147慢病毒表达载体,建立稳定过表达CD147的人肺腺癌A549细胞系,观察过表达CD147后对MMP-9及细胞增殖、侵袭能力的影响.方法 RT-PCR扩增CD147基因全长序列,将序列插入pEGFP载体,构建pEGFP-CD147慢病毒表达载体,随后转入293FT细胞中进行慢病毒包装,用获得的慢病毒毒液感染人肺腺癌细胞系A549,建立稳定过表达CD147的A549细胞系.Real-time PCR检测MMP-9的变化情况,CCK-8及Transwell法检测人肺腺癌细胞增殖、侵袭能力的变化.结果 经限制性内切酶鉴定及测序分析,成功构建了pEGFP-CD 147慢病毒表达载体质粒.Real-time PCR和Western blot检测显示,与对照组相比,转染pEGFP-CD147慢病毒表达载体组的细胞,CD147的表达在mRNA和蛋白两个水平均增高,成功建立了A549-CD147细胞系.上调CD147的表达后,MMP-9的mRNA表达水平明显升高.同时,A549-CD147细胞增殖和侵袭能力明显增加(P<0.05).结论 成功构建CD147慢病毒表达载体和A549-CD147细胞系,过表达CD 147可上调MMP-9的表达,增强人肺腺癌细胞的增殖和侵袭能力.%Background and objective CD 147, a type of transmembrane glycopiotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD 147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD 147 gene was amplified by real-time polymerase chain reaction (RT-PCR), inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable

  11. Expressing Curiosity

    Institute of Scientific and Technical Information of China (English)

    段育付

    2009-01-01

    好奇是人的天性,它有时有助于人们发现或发明新事物。但是在日常交往中,对他人的一些新奇事物或情况则不宜表现出过分的好奇,否则会引起对方的反感。那么,如何用英语表达你的好奇心呢?让我们走进本期话题:Expressing curiosity

  12. Construction of δ-pIRES2-EGFP plasmid and its expression in HEK293 cells%大鼠δ阿片受体基因的pIRES2-EGFP质粒的构建及其在HEK293细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    胡子有; 漆松涛; 张遐; 曹琼; 吴炳义

    2009-01-01

    目的 构建大鼠δ阿片受体基因的pIRES2-EGFP表达质粒,并实现其在HEK293细胞的表达.方法 提取大鼠脑组织总RNA,通过逆转录巢式PCR,扩增δ受体全长cDNA,克隆至pMD20-T载体中,测序鉴定,经酶切、连接克隆人pIRES2-EGFP中,将获得的δ-pIRES2-EGFP重组子转染人HEK293细胞中,应用荧光显微镜观察EGFP和δ基因表达情况.结果 酶切和测序结果表明δ基因正确构建入 pIRES2-EGFP表达质粒中,转染了重组子的HEK293细胞在荧光显微镜下可以观察到绿色荧光,应用细胞免疫荧光,可以观察到δ基因高强度表达.结论 利用巢式RT-PCR等成功构建了δ-pIRES2-EGFP表达质粒,并在HEK293细胞中实现了高效表达.

  13. Establishment of a Stable CHO Cell Line Expressing EGFP Labeled GLUT4%绿色荧光蛋白标记的葡萄糖转运蛋白4稳定表达细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    范俊梅

    2007-01-01

    目的:建立稳定表达EGFP标记的葡萄糖转运蛋白4的CHO细胞系,为研究GLUT4在CHO细胞中的转运调节机制奠定基础.方法:采用分子克隆方法构建GLUT4-EGFP的融合蛋白,在FLP-in的CHO细胞系中表达,潮霉素筛选后得到稳定的细胞系.结果:通过共聚焦显微镜的检测,证明了此稳定细胞系的阳性率达到了99%.定位研究表明大部分GLUT4以囊泡形式分布在CHO细胞胞浆内,但是质膜上也有少量的GLUT4.结论:建立了一个稳定表达GLUT4-EGFP的CHO细胞系,为进一步研究GLUT4的转运提供了一个很好的细胞模型.

  14. Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies.

    Science.gov (United States)

    Bolhassani, Azam; Taheri, Tahereh; Taslimi, Yasaman; Zamanilui, Soheila; Zahedifard, Farnaz; Seyed, Negar; Torkashvand, Fatemeh; Vaziri, Behrouz; Rafati, Sima

    2011-03-01

    Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host-parasite interactions at the cellular level.

  15. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

    Science.gov (United States)

    Xu, Zhen; Chen, Feng; Zhang, Lingling; Lu, Jing; Xu, Peng; Liu, Guang; Xie, Xuemin; Mu, Wenli; Wang, Yajun; Liu, Depei

    2016-10-01

    Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

  16. Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure.

    Science.gov (United States)

    Hanner, Fiona; von Maltzahn, Julia; Maxeiner, Stephan; Toma, Ildiko; Sipos, Arnold; Krüger, Olaf; Willecke, Klaus; Peti-Peterdi, János

    2008-08-01

    Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the

  17. Evaluation of combinatorial cis-regulatory elements for stable gene expression in chicken cells

    Directory of Open Access Journals (Sweden)

    Seo Hee W

    2010-09-01

    Full Text Available Abstract Background Recent successes in biotechnological application of birds are based on their unique physiological traits such as unlimited manipulability onto developing embryos and simple protein constituents of the eggs. However it is not likely that target protein is produced as kinetically expected because various factors affect target gene expression. Although there have been various attempts to minimize the silencing of transgenes, a generalized study that uses multiple cis-acting elements in chicken has not been made. The aim of the present study was to analyze whether various cis-acting elements can help to sustain transgene expression in chicken fibroblasts. Results We investigated the optimal transcriptional regulatory elements for enhancing stable transgene expression in chicken cells. We generated eight constructs that encode enhanced green fluorescent protein (eGFP driven by either CMV or CAG promoters (including the control, containing three types of key regulatory elements: a chicken lysozyme matrix attachment region (cMAR, 5'-DNase I-hypersensitive sites 4 (cHS4, and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE. Then we transformed immortalized chicken embryonic fibroblasts with these constructs by electroporation, and after cells were expanded under G418 selection, analyzed mRNA levels and mean fluorescence intensity (MFI by quantitative real-time PCR and flow cytometry, respectively. We found that the copy number of each construct significantly decreased as the size of the construct increased (R2 = 0.701. A significant model effect was found in the expression level among various constructs in both mRNA and protein (P cis-acting elements decreased the level of gene silencing as well as the coefficient of variance of eGFP-expressing cells (P Conclusions Our current data show that an optimal combination of cis-acting elements and promoters/enhancers for sustaining gene expression in chicken cells

  18. A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells

    Directory of Open Access Journals (Sweden)

    Lejeune Laurence

    2006-11-01

    Full Text Available Abstract Background T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2 promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes. Results First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg. Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 μM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA. This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A. Conclusion These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be

  19. Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays

    Science.gov (United States)

    Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi

    2017-01-01

    Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T1 and T2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T2 progenies and PCR-identified Apr gene segregation in T2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods. PMID:28377783

  20. Analysis of the zebrafish sox9b promoter: Identification of elements that recapitulate organ-specific expression of sox9b.

    Science.gov (United States)

    Burns, Felipe R; Lanham, Kevin A; Xiong, Kong M; Gooding, Alex J; Peterson, Richard E; Heideman, Warren

    2016-03-10

    The SRY-related high-mobility box 9 (SOX9) gene is expressed in many different tissues. To better understand the DNA elements that control tissue-specific expression, we cloned and sequenced a 2.5 kb fragment lying 5' to the zebrafish sox9b gene transcriptional start site. Three regions of this clone contained stable secondary structures that hindered cloning, sequencing, and amplification. This segment and smaller fragmentswere inserted 5' of an EGFP reporter and transgenic fish were raised with the different reporters. Reporter expression was also observed in embryos directly injected with the constructs to transiently express the reporter. Heart expression required only a very short 5' sequence, as a 0.6 kb sox9b fragment produced reporter expression in heart in transgenic zebrafish, and transient experiments showed heart expression from a minimal sox9b promoter region containing a conserved TATA box and an EGR2 element (-74/+29 bp). Reporter expression in transgenic skeletal muscle was consistently lower than in other tissues. Jaw, brain, and notochord expression was strong with the full-length clone, but was dramatically reduced as the size of the fragment driving the reporter decreased from approximately 1.8 to 0.9 kb. The 2.5 kb region 5' of the sox9b contained 7 conserved non-coding elements (CNEs) that included putative hypoxia inducible factor 1α (HIF1α), CAAT box (CCAAT), early growth response protein 2 (EGR2), and core promoter elements. While a synthetic fragment containing all 7 CNEs produced some degree of reporter expression in muscle, jaw, heart and brain, the degree of reporter expression was considerably lower than that produced by the full length clone. These results can account for the tissue-specific expression of sox9b in the developing zebrafish.

  1. Construction of polyoma virus middle T gene vector and its expression in eucaryotic cells%多瘤病毒MT基因真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    何三纲; 赵怡芳; 贾俊

    2001-01-01

    Objective:To construct a plasmid for exploring the role ofpolyoma virus middle T (PyMT) protein in the development of hemangioma. Methods:The open reading frame of PyMT gene from Polyoma virus (Py) genome deleted replication starting sites was digested with restricted enzyme and subcloned into pUC19 plasmid.The resulting plasmid pPyMT was digested with HindⅢ and EcoRI.The PyMT fragment was inserted to plasmid pEGFP1. The recombinant plasmids with proper orientation were identified with analysis of restriction enzymes and PCR.The recombinant vector pPyMT-GFP and the vector-alone pEGFP1 were lined and transfected into mouse skin fibroblasts with electroporation technique.After selected with G418, resistant colonies were obtained. Results:The results showed that the recombinant plasmid could express PyMT efficiently under the control of polyoma virus promoter and enhancer. Conclusions:Because the eukaryotic expression vector pPyMT is derived from the polyoma virus, the PyMT gene could express steadily in mammalian cells.The recombinant vector pPyMT-GFP could be used further to study the effect of PyMT protein on the development of hemangioma.%目的:构建携带多瘤病毒MT癌基因的真核细胞表达载体。方法:从野生型多瘤病毒上取其nt4632至nt1560片段克隆到pUC19上,再将该片段定点插入pEGFP1的HindⅢ和EcoRI位点间,构建携带多瘤病毒MT癌基因的真核表达载体pPyMT-GFP。经线性化,将重组体导入鼠皮肤成纤维细胞中,G418选择培养,得到抗性细胞克隆。结果:构建的pPyMT-GFP质粒在鼠成纤维细胞中有稳定表达。结论:来源于野生型多瘤病毒的PyMT基因在哺乳动物细胞中有稳定表达,其真核表达载体pPyMT-GFP可被进一步用于研究PyMT蛋白在血管瘤发生中的作用。

  2. Prediction and cloning linear Tcell epitopes of P14-3-3 antigen into pEGFP–N1 as a DNA vaccine model to induse immuno response against hydatidosis and it\\'s expression in CHO cell line

    Directory of Open Access Journals (Sweden)

    R mesri

    2015-11-01

    Full Text Available ABSTRACT Background & purpose: Hydatidosis is a zoonotic disease that caused by infection with the larvae of Echinococcus granulosus. Different antigens produced in larval stage of this parasite that recombinant vaccine base these antigens created significant immunity in infected animals. One of the important antigens is p14-3-3 that it's recombinant antigen created considerable immunity in mouse models. In this study according to the high immunity of antigen epitopes region the coding sequence of T-cell epitopes of P14-3-3 was cloned into pEGFP-N1vector in order to produce an effective DNA vaccine model to stimulate high level of Th1 immune response.   Material and method: In this study bioinformatics tools were used to prediction of linear T-Cell epitopes of Echinococcus granulosus P14-3-3 &zeta antigen. The nucleotide coding sequence of these epitopes was synthesized by PCR. the ampliqon was digested with XhoI restriction enzyme and cloned into pEGFP–N1 vector That has been purificated by modified sambrook method with CaCl2 and PEG6000..Positive colony was selected by direct colony PCR and confirmed by the sequencing.and evaluation of it's expression in Eukaryotic cells was done by transformed to CHO cell line with electroporation. Results: Linear T-cell epitopes of Echinococcus granulosus P14-3-3 after prediction,synthesis and amplification wae successfully cloned into pEGFP-N1 vector that purificated by new method with maximum vector and minimum RNA concentration.The expression of new constract in CHO cell line as a eukaryotic cells achivment by fluorescent microscope and will be used as a DNA vaccine model to evaluation immuno response in mouse models.   Discussion: Successfully cloning of The linear T-cell epitppes coding sequence of Echinococcus granulosus P14-3-3&zeta antigen into pEGFP-N1 verificated by sequencing and fluorscent microscope images demonstrated expression of recombinant protein in CHO cell line

  3. 人源脑红蛋白转基因秀丽线虫的制备、鉴定及生理功能分析%Construction, characterization and physiological functions of transgenic C.elegans expressing human neuroglobin

    Institute of Scientific and Technical Information of China (English)

    石锦平; 任长虹; 李媛; 张继业; 范礼斌; 张成岗

    2011-01-01

    目的 以秀丽隐杆线虫为模式生物制备人源脑红蛋白(human neuroglobin,hNgb)转基因线虫,初步探讨外源性hNgb对线虫寿命和产卵率的影响.方法 通过显微注射方法制备hNgb转基因线虫,使用蛋白印迹和免疫组织化学进行鉴定.使用野生型N2和增强型绿色荧光蛋白(EGFP)转基因线虫作为对照组,测量转基因线虫的寿命和产卵率.结果 成功获得hNgb转基因株系线虫,其寿命和产卵率与野生型N2和EGFP转基因线虫相比,差异无统计学意义.结论 外源性hNgb转基因不影响线虫的发育和正常生理功能,可用于Ngb功能的深入研究.%Objective To construct the transgenic Caenorhabditis elegans expressing human neuroglobin(hNgb) and to investigate the effect of hNgb on the brood size and lifespan of C. elegans. Methods The transgenic C. elegans expressing hNgb was obtained with microinjeetion. Expression of hNgb in the transgenic strains was detected by Western blot and immunohistochemistry. The life span and brood size of the C. elegans were detected using wild type N2, while the enhanced green fluorescin protein (EGFP) transgenic strain was used as a control. Results The hNgb transgenic strain was obtained. Compared with wild type C. elegans and EGFP transgenic strain, the transgenic hNgb C. elegans was no statistically significant difference in the life span and brood size. Conclusion The hNgb transgenic strain has no obvious effect on nematode's normal physiological function and could be used for the further functional identification of hNgb.

  4. 表达期特异性双荧光蛋白的转基因弓形虫的构建%Construction of transgenic Toxoplasma gondii expressing dual-fluoresecent stage-specific proteins

    Institute of Scientific and Technical Information of China (English)

    张彦雷; 张厚双; 蔺智兵; 周勇志; 曹杰; 周金林

    2011-01-01

    The conversion from tachyzoite to bradyzoite in Toxoplasma gondii accompany the expression of stage-specific proteins and this experiment aimed at constructing the dual-fluorescent stage-specific parasite. In this experiment, the tachyzoite-specified SAG1 promoter promoted the EGFP (Enhanced Green Fluorescent Protein) gene while the bradyzoite-spec-ified BAG1 promoter promoted the RFP (Red Fluorescent Protein) gene in order to construct the dual-fluorescent expression vector. Then,the vector was transformed to the parasites by electroporation. The results were confirmed by pyrimethimine screening and fluorescence. The PCR assay detected the EGFP gene and RFP gene from the genome of the dual-fluorescent parasites. It was concluded that the Toxoplasma gondii expressing dual-fluoresecent stage-specific proteins were constructed in this experiment.%目的 构建速殖子期特异表达绿色荧光和缓殖子期特异表达红色荧光的转基因弓形虫.方法 利用速殖子期特异性表达的SAG1基因启动子启动绿色荧光蛋白(EGFP)基因,利用缓殖子期特异性表达的BAG1基因启动子启动红色荧光蛋白( RFP)基因,构建期特异性表达双荧光的质粒,通过电转化将质粒转化进弓形虫体内进行表达.结果 经过息疟定抗性筛选得到阳性重组子,经荧光观察观察到了在速殖子时期表达的绿色荧光虫体和经缓殖子诱导后表达红色荧光的缓殖子虫体;通过PCR检测也检测到了EGFP和RFP基因.结论 本试验成功构建表达期特异性双荧光蛋白的转基因弓形虫.

  5. Post-transcriptional regulation of dopamine D1 receptor expression in caudate-putamen of cocaine-sensitized mice.

    Science.gov (United States)

    Tobón, Krishna E; Catuzzi, Jennifer E; Cote, Samantha R; Sonaike, Adenike; Kuzhikandathil, Eldo V

    2015-07-01

    The dopamine D1 receptor is centrally involved in mediating the effects of cocaine and is essential for cocaine-induced locomotor sensitization. Changes in D1 receptor expression have been reported in various models of cocaine addiction; however, the mechanisms that mediate these changes in D1 receptor expression are not well understood. Using preadolescent drd1a-EGFP mice and a binge cocaine treatment protocol we demonstrate that the D1 receptor is post-transcriptionally regulated in the caudate-putamen of cocaine-sensitized animal. While cocaine-sensitized mice express high levels of steady-state D1 receptor mRNA, the expression of D1 receptor protein is not elevated. We determined that the post-transcriptional regulation of D1 receptor mRNA is rapidly attenuated and D1 receptor protein levels increase within 30 min when the sensitized mice are challenged with cocaine. The rapid increase in D1 receptor protein levels requires de novo protein synthesis and correlates with the cocaine-induced hyperlocomotor activity in the cocaine-sensitized mice. The increase in D1 receptor protein levels in the caudate-putamen inversely correlated with the levels of microRNA 142-3p and 382, both of which regulate D1 receptor protein expression. The levels of these two microRNAs decreased significantly within 5 min of cocaine challenge in sensitized mice. The results provide novel insights into the previously unknown rapid kinetics of D1 receptor protein expression which occurs in a time scale that is comparable to the expression of immediate early genes. Furthermore, the results suggest a potential novel role for inherently labile microRNAs in regulating the rapid expression of D1 receptor protein in cocaine-sensitized animals.

  6. Htr2a gene and 5-HT2A receptor expression in the cerebral cortex studied using genetically modified mice

    Directory of Open Access Journals (Sweden)

    Rodrigo Andrade

    2010-08-01

    Full Text Available Serotonin receptors of the 5-HT2A subtype are robustly expressed in the cerebral cortex where they have been implicated in the pathophysiology and therapeutics of mental disorders and the actions of hallucinogens. Much less is known, however, about the specific cell types expressing 5-HT2A receptors in cortex. In the current study we use immunohistochemical and electrophysiological approaches in genetically modified mice to address the expression of the Htr2a gene and 5-HT2A receptors in cortex. We first use an EGFP expressing BAC transgenic mice and identify three main Htr2A gene expressing neuronal populations in cortex. The largest of these cell populations corresponds to layer V pyramidal cells of the anterior cortex, followed by GABAergic interneurons of the middle layers, and nonpyramidal cells of the subplate/Layer VIb. We then use 5-HT2A receptor knockout mice to identify an antibody capable of localizing 5-HT2A receptors in brain and use it to map these receptors. We find strong laminar expression of 5-HT2A receptors in cortex, especially along a diffuse band overlaying layer Va. This band exhibits a strong anteroposterior gradient that closely matches the localization of Htr2A expressing pyramidal cells of layer V. Finally we use electrophysiological and immunohistochemical approaches to show that most, but not all, GABAergic interneurons of the middle layers are parvalbumin expressing Fast-spiking interneurons and that these cells are depolarized and excited by serotonin, most likely through the activation of 5-HT2A receptors. These results clarify and extend our understanding of the cellular distribution of 5-HT2A receptors in the cerebral cortex.

  7. Stable expression of mouse IFN-λ2 in CHO cells and its biological activity analysis%鼠IFN-λ2 CHO细胞系建立及生物学活性的研究

    Institute of Scientific and Technical Information of China (English)

    严玉兰; 袁利学; 刘洋; 曹文雁; 步雪峰; 步志高; 郑金旭

    2010-01-01

    目的 稳定表达鼠IFN-λ2并对其生物学活性进行研究.方法 用水疱口炎病毒(vesicular stomatitis virus,VSV)刺激小鼠脾脏细胞,克隆mIFN-λ2全长基因,构建真核表达载体PCAGG-EGFP-mIFN-λ2,并在CHO细胞稳定表达,且在小鼠黑色素瘤B16细胞上进行抗病毒活性测定;构建MDBK-Mxp-Luc细胞系诱导Mx1抗病毒蛋白产生.结果 pMD18-T-mIFN-λ2双酶切鉴定,出现582 bp大小的条带,成功构建了PCAGG-EGFP-mIFN-λ2真核表达载体;稳定表达mIFN-λ2 CHO的细胞株分泌的上清中mIFN-λ2蛋白在B16细胞上的抗病毒活性为10~4 AU/ml;mIFN-λ2蛋白诱导鼠Mx1抗病毒蛋白的表达,9~12 h达高峰,24 h后消失(P<0.05).结论 建立了稳定表达mIFN-λ2的CHO细胞株,其分泌型mIFN-λ2蛋白具有明显的抗病毒活性,且与诱导Mx1抗病毒蛋白密切相关.%Objective To express mouse IFN-λ2 stably and study its biological activity. Methods Full-length of mIFN-λ2 cDNA was obtained by using RT-PCR from cells of mouse spleen stimulated by ve-sicular stomatitis virus(VSV) and then subcloned to eukaryotic expressing vector PCAGG-EGFP. The recom-binant was transfected into CHO cells. VSV * GFP-B16 system was used to measure the antivirus activity. The constructed cell line MDBK-Mxp-Luc was used to study the character of Mx1 protein induced by the mIFN-λ2. Results The recombinant pMD18-T-mIFN-λ2 was digested by two kinds of enzyme, Sac I and Xho I, to produce the fragment was of 582 bp, and of which the sequence analysis of sequence shows it was entirely consistent with the nucleotide sequences reported in GenBank. PCAGG-EGFP-mIFN-λ2 eukaryotic expressing vector was constructed successfully and expressed stably in CHO cells, and the mRNA of mIFN-λ2 was verified expressing in CHO-PCAGG-EGFP-mIFN-λ2 cell line by RT-PCR. The antivirus activity of in the supernatant secreted by the CHO-PCAGG-EGFP-mIFN-λ2 cell line was 10~4 AU/ml. The mIFN-λ2 pro-tein can could induce the expression of

  8. Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

    Science.gov (United States)

    Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

    2009-05-27

    Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia.

  9. Expression of Two N1 Clones with Single Amino Acid Dissimilarity of Avian Influenza H5N1 Virus

    Directory of Open Access Journals (Sweden)

    RISZA HARTAWAN

    2012-12-01

    Full Text Available Two clones of N1 gene derived from isolate A/Dk/Tangerang/Bbalitvet-ACIAR-TE11/2007 (H5N1 exhibit single mismatch of amino acid sequence at position 242 that is threonine and methionine for the clone #3 and #5, respectively. In order to evaluate the effect of the amino acid substitution, these clones were inserted into two different expression vectors that are pEGFP-C1 and pcDNA-3.3 TOPO® TA cloning. Subsequently, the respective recombinant clones were transfected into eukaryotic cells, including CEF, RK13 and VERO using Lipofectamine ‘plus’ reagent. As a result, the clone #3 retaining atypical sequence showed lower expression level rather than the clone #15 in both vectors and all type of cells. The 3D conformational modelling revealed that the mutation occurs in the inner part of glycoprotein embedded within envelope or matrix. Therefore, the missense mutation seems has no effect on the antigenic properties of neuraminidase but this substitution by any means causes lethal mutagenesis in the individual gene expression by reducing level of protein transcript.

  10. Hyperthermia Differently Affects Connexin43 Expression and Gap Junction Permeability in Skeletal Myoblasts and HeLa Cells

    Directory of Open Access Journals (Sweden)

    Ieva Antanavičiūtė

    2014-01-01

    Full Text Available Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK in hyperthermia-induced changes of connexin43 (Cx43 expression and permeability of Cx43 gap junctions (GJs in the rabbit skeletal myoblasts (SkMs and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC. In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  11. Hyperthermia differently affects connexin43 expression and gap junction permeability in skeletal myoblasts and HeLa cells.

    Science.gov (United States)

    Antanavičiūtė, Ieva; Mildažienė, Vida; Stankevičius, Edgaras; Herdegen, Thomas; Skeberdis, Vytenis Arvydas

    2014-01-01

    Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK) in hyperthermia-induced changes of connexin43 (Cx43) expression and permeability of Cx43 gap junctions (GJs) in the rabbit skeletal myoblasts (SkMs) and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h) enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC). In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  12. Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification.

    Science.gov (United States)

    Betts, Zeynep; Dickson, Alan J

    2016-03-01

    The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.

  13. drd2-cre:ribotag mouse line unravels the possible diversity of dopamine d2 receptor-expressing cells of the dorsal mouse hippocampus.

    Science.gov (United States)

    Puighermanal, Emma; Biever, Anne; Espallergues, Julie; Gangarossa, Giuseppe; De Bundel, Dimitri; Valjent, Emmanuel

    2015-07-01

    Increasing evidences suggest that dopamine facilitates the encoding of novel memories by the hippocampus. However, the role of dopamine D2 receptors (D2R) in such regulations remains elusive due to the lack of the precise identification of hippocampal D2R-expressing cells. To address this issue, mice expressing the ribosomal protein Rpl22 tagged with the hemagglutinin (HA) epitope were crossed with Drd2-Cre mice allowing the selective expression of HA in D2R-containing cells (Drd2-Cre:RiboTag mice). This new transgenic model revealed a more widespread pattern of D2R-expressing cells identified by HA immunoreactivity than the one initially reported in Drd2-EGFP mice, in which the hilar mossy cells were the main neuronal population detectable. In Drd2-Cre:RiboTag mice, scattered HA/GAD67-positive neurons were detected throughout the CA1/CA3 subfields, being preferentially localized in stratum oriens and stratum lacunosum-moleculare. At the cellular level, HA-labeled cells located in CA1/CA3 subfields co-localized with calcium-binding proteins (parvalbumin, calbindin, and calretinin), neuropeptides (neuropeptide Y, somatostatin), and other markers (neuronal nitric oxide synthase, mGluR1α, reelin, coupTFII, and potassium channel-interacting protein 1). These results suggest that in addition to the glutamatergic hilar mossy cells, D2R-expressing cells constitute a subpopulation of GABAergic hippocampal interneurons.

  14. The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies

    Directory of Open Access Journals (Sweden)

    Burt Austin

    2009-07-01

    Full Text Available Abstract Background Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae. Results We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae. Conclusion We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

  15. Expression ofpprI andpprA genes from deinococcus radiodurans in eukaryotic 293T cells%耐辐射奇球菌pprI和pprA基因在真核细胞293T中的表达

    Institute of Scientific and Technical Information of China (English)

    肖潇; 马云; 肖方竹; 唐艳; 杨奇; 黄波; 唐旻; 何淑雅

    2016-01-01

    To construct fluorescence expression plasmids pEGFP-N1-pprA and pDsRed1-N1-flag-pprI, pGADT-7-pprA and pet28a-pprI plasmid constructed at an earlier laboratory stage was used as the template, and Lipofectamine 2000 was employed to transfect two recombinant vectors into 293T cells. A fluorescent microscope was used for Fluorescence observation, and Western blot was employed to examine the expression of the fusion protein. Double digestions and the agarose gel electrophoresis showed that target bands appeared at 4700 bp and 1000 bp, 4800 bp and 1100 bp. No apparent frameshift mutation occurred as shown in the sequencing results. The fluorescent microscopy showed red and green phosphors; the Western blot results indicated protein expression at 65 kDa and 60 kDa. pDsRed1-N1-flag-pprI and pEGFP-N1-pprA were successfully constructed to express proteins for eukaryotic cells 293T in vitro. The results indicated that prokaryotic genespprA and pprI could co-express proteins in eukaryotic cells successfully, and laid a foundation for the interaction and synergism ofpprA,pprI, and their products in the regulation network of radiation verified by DR, enhancing the radiation resistance of eukaryotic cells.%以pGADT-7-pprA、pet28a-pprI载体为模板,构建pEGFP-N1-pprA、pDsRed1-N1-flag-pprI真核表达载体,脂质体2000介导将两个重组载体共转入293T细胞.双酶切及琼脂糖凝胶电泳显示在4700、1000 bp处与4800、1100 bp处出现目的条带.测序结果显示构建序列与模板序列一致,氨基酸序列100%正确.荧光显微镜下见到红色和绿色荧光;Western blot结果显示在不同检测水平65 kDa及60 kDa大小处有融合蛋白表达.结果提示pEGFP-N1-pprA、pDsRed1-N1-flag-pprI真核表达载体构建成功,并在离体293T细胞中共同表达蛋白.证明原核基因pprI、pprA能够在真核细胞中共表达,为后续实验验证DR菌高抗性基因pprA、pprI及其产物在辐射调控网络中的相互作用和协同作用、

  16. mTEL-cFms激酶结构域融合蛋白真核表达载体的构建及其对信号转导和转录激活因子核转位的影响%Establishment of mTEL-cFmskd eukaryotic expression vector and its effect on STAT1/3 nuclear translocation

    Institute of Scientific and Technical Information of China (English)

    杨胜乾; 龙隆; 李微; 王莉莉

    2011-01-01

    OBJECTIVE To construct a eukaryotic expression vector of mTEL-cFmskd and study its effect on nuclear translocation of the signal transducer and activator of transcription 1/3 ( STAT1/3 ). METHODS By recombinant DNA technology, the DNA sequence of polypeptide for myristoylation of human c-Src, helix-loop-helix domain of human TEL, kinase domain of macro-phage colony-stimulating factor receptor and c-Myc tag were inserted into pCORON/neo plasmid to generate pCORON/neo-HcSrc-Tel-cfmskd-Myc eukaryotic expression vector. The mTEL-cFmskd expression vector pCORON/neo-HcSrc-Tel-cfmskd-Myc and the c-Fms expression vector pCORON/ neo-cfms were transfected into U2OS ( expressing GFP-STAT1 ) and BHK ( expressing EGFP-STAT3) cells. After 24 h, the cells were fixed, stained and then imaged on the IN Cell Analyzer 1000. The image was analyzed using the Nuclear Trafficking Analysis Module. RESULTS Restriction enzyme digestion and plasmid sequencing confirmed the successful construction of pCORON/neo-HcSrc-Tel-cfmskd-Myc plasmid. mTEL-cFmskd was expressed in cells, and caused nuclear translocation of GFP-STAT1 and EGFP-STAT3 24 h after transfection. C-Fms inhibitors GW2580 and Sutent could block the nuclear translocation of EGFP-STAT3 by mTEL-cFmskd. CONCLUSION mTEL-cFmskd expression vector pCORON/neo-HcSrc-Tel-cfmskd-Myc is successfully constructed and functional mTEL-cFmskd is expressed in GFP-STAT1_U2OS and EGFP-STAT3_BHK cells.%目的 构建激酶盘真核表达载体,观察豆蔻酰化的TEL转录调节因子HLH结构域与c-Fms激酶结构域融合蛋白( mTEL-cFmskd)的表达对信号转导和转录激活因子1(STAT1)和STAT3核转位的影响.方法 利用DNA重组技术,将人的c-Src豆蔻酰化多肽、TEL转录调节因子HLH结构域、c-Fms激酶结构域以及c-Myc标签的DNA序列克隆在pCORON/neo载体上,构建pCORON/neo-HcSrc-Tel-cfmskd-Myc真核表达载体.将载体转染至稳定表达GFP-STAT1的人骨肉瘤细胞(U2OS)和稳定表达EGFP-STAT3

  17. Generation and characterization of blood vessel specific EGFP transgenic zebrafish via Tol2 transposon mediated enhancer trap screen%利用Tol2转座子介导的增强子诱捕技术获得血管相关转基因斑马鱼系

    Institute of Scientific and Technical Information of China (English)

    薛峪霖; 肖安; 文路; 贾岳; 高岳; 朱作言; 林硕; 张博

    2010-01-01

    心血管系统形成于胚胎发育极早期并为其他器官的发育、维持、修复所必需,血管生长异常可造成多种疾病.然而,由于研究对象所限,胚胎血管的发育机制尚未完全阐明,调控血管发育的基因也所知有限.通过Tol2转座子介导的大规模增强子诱捕筛选到26个血管特异表达绿色荧光蛋白(EGFP)报告基因的转基因斑马鱼系,其中有一些品系在胚胎的某些特异血管结构中表达绿色荧光.通过linker-mediated PCR克隆到22个鱼系中Tol2插入位点附近的斑马鱼基因组序列,其中有17个鱼系的Tol2插入可定位到现有的斑马鱼基因组中的单一位点.通过整体胚胎原位杂交对插入位点附近的基因进行表达谱分析,得到8个表达谱与转基因鱼系一致的基因,涵盖了9个鱼系,其中dusp5基因对应于2个不同的鱼系.这8个基因中包括hhex、ets1a和dusp5等3个功能已知的基因,但是大部分(5个)基因在斑马鱼中尚无功能研究,分别为zvsg1、micall2a、arl8b(1 of 2)、zgc:73355以及hecw2(1 of 2).hhex和ets1a基因对血管与血细胞前体的发育具有重要作用,所获得的EGFP报告基因受hhex或ets1a基因增强子控制的转基因斑马鱼(mp378b和mp430c-2)为国际首例,为深入研究这两个基因在血管与血液发育中的作用机制提供了新的机遇.筛选到的功能未知基因可以用来进一步研究其在血管发育中的功能;同时,利用所获得的转基因鱼系,可以实现实时、动态观察成血管细胞的起源、分化与基因表达调控,并可用于高通量小分子药物筛选等重要研究.

  18. Construction of Fibroblast Growth Factor 5 Gene (FGF5) Hair Follicle Specific Expression Vector and Transfected into Cashmere Goat (Capra hircus) Fetal Fibroblast Cells%绒山羊成纤维细胞生长因子5基因(FGF5)毛囊特异性表达载体的构建及转染胎儿成纤维细胞

    Institute of Scientific and Technical Information of China (English)

    康健; 胡广东; 权富生; 张涌

    2014-01-01

    The quality and yield of cashmere goat wool are closely related to the growth and development of skin hair follicles. This study aimed to construct an eukaryotic expression vector and prove whether it could specifically express fibroblast growth factor 5 gene (FGF5) in the hair follicles of cashmere goat (Capra hircus). The keratin associated protein 6-1 (KAP6-1) promoter and FGF5 gene coding sequence were obtained by PCR. The vector pEGFP-N1-KF, a hair follicles specific expression vector of FGF5, was constructed by inserting the KAP6-1 promoter into the CMV promoter-less pEGFP-N1, and then connecting FGF5 to the downstream of KAP6-1 promoter, using Porcine teschovirus 2A peptide (P2A) to link FGF5 and enhanced green fluorescent protein (EGFP) gene. The constructed vector was transfected into cashmere goat fetal fibroblast cells and two methods were used to verify its functionality including qRT-PCR and Western blot. The enzyme digestion results showed the vector was correctly constructed. The qRT-PCR results indicated that the FGF5 gene could express in fetal fibroblast cells. The Western blotting results confirmed that the fusion protein could be availably spelited into FGF5 and GFP protein by the functionality of P2A. The result showed that the vector pEGFP-N1-KF which could specifically express FGF5 gene was successfully constructed and could normally expressed in cashmere goat fetal fibroblast cells.%绒山羊的绒毛品质、产量与皮肤毛囊的生长发育密切相关。本研究旨在构建绒山羊(Capra hircus)成纤维细胞生长因子5基因(fibroblast growth factor 5, FGF5)的毛囊特异性表达载体,并证明其表达的有效性。分别以绒山羊基因组和cDNA为模板,利用PCR方法克隆角蛋白关联蛋白6-1(keratin associated protein 6-1, KAP6-1)基因的启动子和FGF5基因的编码区序列(coding sequence, CDS),并将这两个元件连接到去除CMV启动子的真核表达载体pEGFP-N1上,FGF5

  19. Growth and activation of PI-3K/PKB and Akt by stromal cell-derived factor 1α in endometrial carcinoma cells with expression of suppressor endoprotein PTEN

    Institute of Scientific and Technical Information of China (English)

    LI Xiao-ping; ZHAO Dan; GAO Min; ZHAO Chao; WANG Jian-liu; WEI Li-hui

    2006-01-01

    Background Mutation or deletion in the phosphatase and tensin homologue deleted on chromosome ten (PTEN)gene has been identified as an important cause of endometrial carcinoma; stromal cell derived factor-1α (SDF-1α)exerts growth-promoting effects on endometrial cancer cells through activation of the PI-3 kinase/Akt pathway and downstream effectors such as extracellular-responsive kinase (ERK). In this study, a plasmid containing the PTEN gene was transfected into Ishikawa cells to investigate the difference in growth and signal transduction between Ishikawa-PTEN and Ishikawa cells after SDF-1α stimulation, and to study mechanisms of the involvement of PTEN protein in endometrial carcinoma development.Methods Ishikawa cells were transfected with a plasmid (pLXSN-PTEN) containing the PTEN gene and a plasmid (pLXSN-EGFP) with enhanced green fluorescent protein (EGFP). Cells were then screened to obtain Ishikawa-PTEN cells and Ishikawa-neo cells that can both stably express PTEN protein and EGFP. Expression of PTEN protein, phosphorylation levels of AKT and ERK (pAKT and pERK) and growth differences in Ishikawa-PTEN, Ishikawa-neo and Ishikawa cells before and after SDF-1α stimulation were then determined by Western blots and MTT assays.Results Western blot analysis showed that Ishikawa cells produced PTEN after transfection with the PTEN gene. At 15 minutes after SDF-1α stimulation, the pAKT level of Ishikawa-PTEN cells was lower than that of Ishikawa-neo cells and Ishikawa cells. There was no significant difference in pERK levels among the three cell lines. The positive effect of SDF-1α on Ishikawa-PTEN cells growth was markedly less than the effect on Ishikawa-neo and Ishikawa cells. However, in the absence of SDF-1α stimulation (baseline), the pAKT level in Ishikawa-PTEN cells was less than that in Ishikawa cells. There was a significant difference in growth between the Ishikawa-PTEN cells and the Ishikawa-neo cells.Conclusions PTEN gene transfection can

  20. Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors.

    Science.gov (United States)

    Faust, Georg; Janzen, Nils H; Bendig, Christoph; Römer, Lin; Kaufmann, Klaus; Weuster-Botz, Dirk

    2014-10-01

    Miniature bioreactors under parallel fed-batch operations are not only useful screening tools for bioprocess development but also provide a suitable basis for eventual scale-up. In this study, three feeding strategies were investigated: besides the established intermittent feeding by a liquid handler, an optimized microfluidic device and a new enzymatic release system were applied for parallel fed-batch cultivation of Escherichia coli HMS174(DE3) and BL21(DE3) strains in stirred-tank bioreactors on a 10 mL scale. Lower fluctuation in dissolved oxygen (DO) and higher optical densities were measured in fed-batch processes applying the microfluidic device or the enzymatic glucose/fructose release system (conversion of intermittently added sucrose by an invertase), but no difference in dry cell weights (DCW) were observed. With all three feeding strategies high cell densities were realized on a milliliter scale with final optical density measured at 600 nm (OD600 ) of 114-133 and final DCW concentrations of 69-70 g L(-1) . The effect of feeding strategies on the expression of two heterologous proteins was investigated. Whereas no impact was observed on the expression of the spider silk protein eADF4(C16), the fluorescence of enhanced green fluorescence protein (eGFP) was reproducibly lower, if an intermittent glucose feed was applied. Thus, the impact of feeding strategy on expression is strongly dependent on the E. coli strain and/or expressed protein. As a completely continuous feed supply is difficult to realize in miniature bioreactors, the enzymatic release approach from this study can be easily applied in all microfluidic system to reduce fluctuations of glucose supply and DO concentrations.

  1. Prokaryotic expression and subcellular localization of Japanese encephalitis virus NS5 protein%乙型脑炎病毒NS5蛋白原核表达与亚细胞定位研究

    Institute of Scientific and Technical Information of China (English)

    栗永茂; 曹胜波; 陈龙; 霍雨艳; 熊涛; 吴光旭

    2011-01-01

    The NS5 gene of Japanese encephalitis virus was amplified by PCR and cloned into the prokaryotic expression vector pET-28a. The recombinant NS5 protein was expressed highly under induction of IPTG in the E. Coli. Using BALB/c female mice immuned with the purified recombinant NS5 protein, NS5 protein-specific polyclonal antibody were prepared. Meanwhile, BHK-21 cells were transfected with eukaryotic expression plasmid pcDNA-NS5-EGFP of NS5 gene and green fluorescent protein (EGFP) fusion expression. Finally, the subcellular localization of NS5 protein in BHK-21 cells was observed by using laser confocal fluorescence microscope. The result indicates that the protein particles are irregularly distributed in the whole cell and particularly in the cytoplasm.%目的 对乙型脑炎病毒NS5蛋白进行原核表达和亚细胞定位研究.方法 通过PCR扩增乙型脑炎病毒NS5基因,并将之克隆至原核表达载体pET-28a(+)中,构建的重组表达载体转化大肠杆菌感受态细胞,经IPTG诱导表达目的蛋白.将表达产物纯化后,免疫BALB/c小鼠,制备NS5蛋白特异性的多克隆抗体.同时,构建NS5基因与绿色荧光蛋白(EGFP)融合表达的真核表达质粒pcDNA-NS5-EGFP,将之转染BHK-21细胞,利用激光共聚焦荧光显微镜观察NS5蛋白在BHK-21细胞中的亚细胞定位.结果 NS5蛋白在大肠杆菌中获得了高效表达,表达蛋白分子量大小约为103kD,该蛋白在真核细胞中的表达呈颗粒状不均匀分布于整个细胞,且颗粒状主要分布在细胞质中.结论 成功表达乙型脑炎病毒NS5蛋白,并对NS5蛋白的亚细胞定位进行了分析,为进一步研究NS5蛋白提供依据.

  2. Multigene expression in stable CHO cell pools generated with the piggyBac transposon system.

    Science.gov (United States)

    Balasubramanian, Sowmya; Wurm, Florian M; Hacker, David L

    2016-09-01

    Heterogenous populations of recombinant cells (cell pools) stably expressing 1-4 transgenes were generated from Chinese hamster overy (CHO) cells with the piggyBac (PB) transposon system. The cell pools produced different combinations of three model proteins-enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody. Each transgene was present on a separate PB donor plasmid with either the same or a different selection gene. In both cases, we obtained PB-derived cell pools with higher recombinant protein yields than from cell pools generated by conventional gene delivery. In PB-derived cell pools generated using a single selection agent, both protein production and the number of integrated copies of each transgene declined as the number of transfected transgenes increased. However, the total number of integrated transgenes was similar regardless of the number of different transgenes transfected. For PB-derived cell pools generated by selection of each transgene with a different selection agent, the total number of integrated transgenes increased with the number of transfected transgenes. The results suggest that the generation of cell pools producing multiple recombinant proteins is feasible and that the method is more efficient when each individual transgene is selected with a different marker. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1308-1317, 2016.

  3. Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats

    Directory of Open Access Journals (Sweden)

    Janssen William GM

    2006-01-01

    Full Text Available Abstract Background Intrathecal (IT gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV, one of the most promising vector types for clinical use. Results Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. Conclusion sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2

  4. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    Directory of Open Access Journals (Sweden)

    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  5. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

    Directory of Open Access Journals (Sweden)

    Lee Okhyun

    2012-06-01

    Full Text Available Abstract Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38 in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff, contains three copies of oestrogen response elements (3ERE that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein. Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2, the synthetic oestrogen 17α- ethinyloestradiol (EE2, and the relatively weak environmental oestrogen nonylphenol (NP, and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures. For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish. We

  6. Recombinant Pseudorabies Virus (PRV Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds

    Directory of Open Access Journals (Sweden)

    Yan-Dong Tang

    2016-03-01

    Full Text Available A Pseudorabies virus (PRV variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR/Cas9 technology. However, identification of single guide RNA (sgRNA through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.

  7. Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells.

    Science.gov (United States)

    Mariati; Yeo, Jessna H M; Koh, Esther Y C; Ho, Steven C L; Yang, Yuansheng

    2014-01-01

    The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation.

  8. A practical method of expression, purification and identification of beta-site app-cleaving enzyme%一种实用的β-分泌酶表达、纯化和活性鉴定方法

    Institute of Scientific and Technical Information of China (English)

    朱瑞; 伍巧燕; 张兴梅; 高小芳; 卫佩如; 张馨宇; 王方

    2015-01-01

    Objective To introduce a practical method that can be used to efficiently express,purify and identify Alzheimer's disease (AD) related beta-site app-cleaving enzyme 1 (BACE1) in common eukaryotic cells.Methods BACE1 cDNA was fished out from human brain cDNA library and ligated into the pEGFP-c3 expression vector,and then,the recombinant plasmid was transfected into the HEK293 cells.The BACE1 protein was purified with TALON Mental Affinity Resins column.The target protein was identified by Western blotting and fluorescence resonance energy transfer (FRET).BACE1 Activity Assay Kit was employed to test the activity of purified BACE1 in vitro.The recombinant BACE1/pEGFP-c3 plasmid and amyloid precusor protein (APP)/pDsRed-Monomer-N1 plasmid were co-transfected to the HEK293 cells and the cleavage activity of BACE1 in the cells was identified by Western blotting.Results The sequencing data of the obtained BACE1 gene were identical with those in GenBank.Activity test showed that the fluorescent values of blank controls,expressed BACE1 and standard BACE1 were 55.013±3.597,1836.629±154.195 (n=3) and 2639.548±207.1901 (n=3),respectively;as compared with the control group,significant differences were noted in both of the two groups (F=78.681,P=0.000);however,there is no significant difference between expressed BACE1 and standard BACE1 groups (P>0.05).Westem blotting showed the co-transfected BACE1 could cleave APP in HEK293 cells and the CTF-APP band was detectable.Conclusion A practical protocol is established for high expression,purification and identification of BACE1 in HEK293 cells,which is helpful to obtain BACE1,an important molecular target in AD research and treatment.%目的 探讨一种在常用真核细胞系中表达、纯化和鉴定阿尔茨海默病(AD)相关蛋白-β-分泌酶(BACE1)的方法. 方法 在从人脑基因库中获取BA CE1序列并成功扩增的基础上,将其插入带有增强绿色荧光蛋白(EGFP)的表达载体pEGFP-c3中.将BACE1/pEGFP

  9. Pineal gland expression of the transcription factor Egr-1 is restricted to a population of glia that are distinct from nestin-immunoreactive cells.

    Science.gov (United States)

    Man, Pui-Sin; Carter, David A

    2008-02-01

    Egr-1 is a plasticity-related transcription factor that has been implicated in circadian regulation of the pineal gland. In the present study we have investigated the cellular expression pattern of Egr-1 in the adult rat pineal. Egr-1 protein is restricted to the nucleus of a sub-population of cells. These cells were characterised using a new transgenic rat model (egr-1-d2EGFP) in which green fluorescent protein is driven by the egr-1 promoter. Cellular filling by GFP revealed that Egr-1-positive cells exhibited processes, indicating a glial cell-type morphology. This was confirmed by co-localizing the GFP-filled processes with vimentin and S-100beta. However, GFP/Egr-1 is expressed in only a tiny minority of the previously identified Id-1/vimentin-positive glial cells and therefore represents a novel sub-set of this (GFAP-negative) glial population. We have also demonstrated for the first time an extensive network of nestin-positive cells throughout the adult pineal gland, however these cells do not co-express Egr-1. Our studies have therefore broadened our understanding of the cell populations that constitute the adult pineal. Cellular localization of Egr-1 has revealed that this factor does not appear to be directly involved in pinealocyte production of melatonin but is required in a sub-set of pineal glia.

  10. AAV-mediated expression of BAG1 and ROCK2-shRNA promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury.

    Science.gov (United States)

    Challagundla, Malleswari; Koch, Jan Christoph; Ribas, Vinicius Toledo; Michel, Uwe; Kügler, Sebastian; Ostendorf, Thomas; Bradke, Frank; Müller, Hans Werner; Bähr, Mathias; Lingor, Paul

    2015-07-01

    A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno-associated virus (AAV)-mediated over-expression of BAG1 and ROCK2-shRNA in the red nucleus to trace [by co-expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein over-expression versus AAV.shRNA-mediated down-regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.

  11. Cloning of Brandt's vole oxytocin gene and its over-expression after cell transfection on lentiviral vector%布氏田鼠催产素基因的克隆及通过慢病毒载体转染细胞后的过表达检测

    Institute of Scientific and Technical Information of China (English)

    周文君; 施海霞; 宋铭晶

    2013-01-01

    目的:构建表达布氏田鼠催产素基因的慢病毒载体及过表达检测.方法:从布氏田鼠脑组织提取RNA逆转录PCR得到OT的cDNA片段,并分别连接到带有荧光素酶(luciferase,LUC)标记的慢病毒载体PGK启动子下游,以及带有绿色荧光蛋白(EGFP)标记的EF1A启动子下游,通过这两个载体转染细胞,分别利用生物发光标记与荧光标记这两种技术示踪OT基因的表达;再通过提取RNA逆转录以及实时荧光定量PCR检测OT基因不同模板转录的拷贝数.结果:成功构建LUC标记和EGFP标记的表达布氏田鼠催产素基因的慢病毒载体,进行了真核表达检测,并建立了实时荧光定量PCR的方法.结论:催产素(oxytocin,OT)基因是哺乳动物特有的神经垂体激素,与社会认知行为和社会适应行为有关,为研究该基因的功能奠定基础.%Objective:To construct lentiviral vector of OT gene expression in Brandt's vole and detect the expression quantity and expressed area of OT gene.Methods:Total RNA extracted from brain tissue of Brandt's vole was amplified by RT-PCR for the cDNA fragments of OT gene and connected to two kinds of lentiviral vector,down stream of PGK or EF1A respectively.The two kinds of recombinant plasmid with OT gene labeling of luciferase,and eGFP marker respectivly,were transfected into 293T cells.The OT gene expression was detected in vivo by fluorescent imaging of luciferase or eGFP marker.At the same time,SYBR Green Ⅰ real-time RT-PCR system was established to detect the copy number of OT gene in different samples.Results:The lentiviral vector labeled by LUC and EGFP was successfully constructed to express oxytocin gene of Brandt's vole,and real-time fluorescent PCR method was also established to detect the copy numbers of OT gene in transgenic cells.Conclusion:Oxytocin gene is a peculiar neurohypophysial hormone of mammal,associated with social cognition behaviors and social adaption behaviors.

  12. Recognition of environmental estrogen-like compounds based on fluorescent imaging analysis of ERα-EGFP nuclear granules formation%基于ERα-EGFP核颗粒形成的荧光成像分析快速识别环境中雌激素样物质

    Institute of Scientific and Technical Information of China (English)

    冯帆; 王莉莉

    2015-01-01

    目的:利用基于荧光成像分析的细胞高内涵分析( high content analysis ,HCA)技术,建立快速识别环境中雌激素样物质的方法。方法以稳定表达雌激素受体α( ERα)-增强型绿色荧光蛋白( enhanced green fluorescent protein,EGFP)融合蛋白(ERα-EGFP)的人骨肉瘤U2OS细胞为模型,以雌激素(17-β-雌二醇)为阳性对照药,以黄体酮为阴性对照药,基于ERα-EGFP核颗粒形成的荧光成像分析探查环境污染物质双酚A、壬基酚、邻苯二胺、对氨基苯酚、间苯二酚、间氨基苯酚、对苯二胺和甲基-2,5-二胺硫酸盐等对ERα-EGFP细胞核颗粒形成作用( Foci作用)的影响,分析其量效关系;并利用荧光素酶报告基因实验验证HCA的结果。结果 HCA结果显示,仅有双酚A、壬基酚和阳性药17-β-雌二醇能够剂量依赖地促进ERα-EGFP 细胞核颗粒形成作用,其EC50分别为(1.48±1.79)μmol/L、(3.70±0.78)μmol/L及(4.17±0.41) nmol/L,最小检出浓度分别为1 nmol/L(17-β-雌二醇)、300 nmol/L(双酚A和壬基酚);邻苯二胺、对氨基苯酚、间苯二酚、间氨基苯酚、对苯二胺和甲基-2,5-二胺硫酸盐等不能诱导ERα-EGFP细胞核颗粒形成作用。荧光素酶报告基因实验结果同样显示,活性药物双酚A、壬基酚和阳性药17-β-雌二醇能够剂量依赖地诱导ERα报告基因ERE-Luc的活性,其EC50分别为(2.31±0.21)μmol/L、(6.60±0.94)μmol/L及(4.46±0.56)nmol/L;最小检出浓度是3 nmol/L(17-β-雌二醇)、300 nmol/L(双酚A)和1μmol/L (壬基酚)。 HCA和荧光素酶报告基因实验的结果基本一致,但HCA方法更为灵敏,实验步骤更为简便。结论基于荧光成像定量分析的HCA技术能够快速和灵敏地识别具有ERα激动活性的环境物质,是分析环境雌激素的有效方法。受试

  13. Express web application development

    CERN Document Server

    Yaapa, Hage

    2013-01-01

    Express Web Application Development is a practical introduction to learning about Express. Each chapter introduces you to a different area of Express, using screenshots and examples to get you up and running as quickly as possible.If you are looking to use Express to build your next web application, ""Express Web Application Development"" will help you get started and take you right through to Express' advanced features. You will need to have an intermediate knowledge of JavaScript to get the most out of this book.

  14. Stem cells with FGF4-bFGF fused gene enhances the expression of bFGF and improves myocardial repair in rats

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiang-Qi; Chen, Liang-Long, E-mail: xhzlyx@126.com; Fan, Lin; Fang, Jun; Chen, Zhao-Yang; Li, Wei-Wei

    2014-04-25

    Highlights: • BFGF exists only in the cytoplasm of live cells. • BFGF cannot be secreted into the extracellular space to promote cell growth. • We combine the secretion-promoting signal peptide of FGF4. • We successfully modified BMSCs with the fused genes of FGF4-bFGF. • We promoted the therapeutic effects of transplanted BMSCs in myocardial infarction. - Abstract: The aim of this study was to investigate whether the modification of bone marrow-derived mesenchymal stem cells (BMSCs) with the fused FGF4 (fibroblast growth factor 4)-bFGF (basic fibroblast growth factor) gene could improve the expression and secretion of BFGF, and increase the efficacies in repairing infarcted myocardium. We used In-Fusion technique to construct recombinant lentiviral vectors containing the individual gene of bFGF, enhanced green fluorescent protein (EGFP), or genes of FGF4-bFGF and EGFP, and then transfected these lentiviruses into rat BMSCs. We conducted an in vitro experiment to compare the secretion of bFGF in BMSCs infected by these lentiviruses and also examined their therapeutic effects in the treatment of myocardial infraction in a rodent study. Sixty rats were tested in the following five conditions: Group-SHAM received only sham operation as controls; Group-AMI received only injection of placebo PBS buffer; Group-BMSC, Group-bFGF and Group-FGF4-bFGF received implantation of BMSCs with empty lentivirus, bFGF lentivirus, and FGF4-bFGF lentivirus, respectively. Our results found out that the transplanted FGF4-bFGF BMSCs had the highest survival rate, and also the highest myocardial expression of bFGF and microvascular density as evidenced by Western blotting and immunohistochemistry, respectively. As compared to other groups, the Group-FGF4-BFGF rats had the lowest myocardial fibrotic fraction, and the highest left ventricular ejection fraction. These results suggest that the modification of BMSCs with the FGF4-bFGF fused gene can not only increase the expression of

  15. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal

    Directory of Open Access Journals (Sweden)

    Macmaster Suzanne

    2002-06-01

    Full Text Available Abstract Background Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes 1. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP in embryonic stem (ES cells and mice 2. Results In this study we have used embryonic stem (ES cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP and enhanced yellow fluorescent protein (EYFP, two mutant and spectrally distinct color variants of wild type (wt GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid diploid and polarized (tetraploid diploid chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal. Conclusions GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or

  16. Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

    LENUS (Irish Health Repository)

    Andrews, E J

    2012-02-03

    BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.

  17. Gene Clone, Subcellular Localization of Expression Products of H-FABP and the Preparation of Transgenic Mice in Xuhuai Goat%徐淮山羊H-FABP基因克隆、表达产物亚细胞定位的研究及转基因小鼠的制备

    Institute of Scientific and Technical Information of China (English)

    阴彦辉; 韦光辉; 李伟; 朱才业; 张亚妮; 杜立新; 曹文广; 李碧春

    2012-01-01

    The purpose of this study was to clone heart-type fatty acid binding protein (H-FABP) gene cDNA of Xuhuai goat, and to explore its bioinformatics function and the possibility of preparation of transgenic animals among heterogeneous species. The subcelluar location H-FABP was detected by EGFP fusion protein and its expression was observed in vitro. Reverse transcription PCR (RT-PCR) technology was used to clone the H-FABP gene cDNA of Xuhuai goat, its biological information characteristics was analyzed by online software, then the expression vector pEGFP-H-FABP was constructed. The transfection of goat fibroblasts (GEF) was performed by Liposomes (LTX), and fluorescence was observed under inverted microscope after 48 h. The RT-PCR was conducted to detect mRNA expression of H-FABP in GEF. The pEGFP-H-FABP was injected into mouse testicular and its expression was detected at the level of DNA and protein. The complete CDS size of H-FABP was 402 bp, encoding 133 amino acids with GenBank accession number (AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding regions of human, chicken, brown rat, cow, wild boar, donkey and zebra fish, the similarity was 89% , 76%, 85% , 84% , 93% , 91% , 70% , respectively, amino acid sequence homol-ogy was 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. The signal peptide was not found in H-FABP protein. The RT-PCR results showed the H-FABP mRNA expressed successfully in vitro. pEGFP-H-FABP was successfully constructed, and H-FABP mRNA was expressed. The H-FABP protein was localized in the cytoplasm which was in line with the result of online prediction. The gene can aslo be expressed in mice transiently and persistencely after intravenous and testicular injection. The H-FABP gene cDNA of Xuhuai goat was cloned successfully, and it was conservative during the evolutionary process, there was no signal peptide in protein. The H-FABP protein was located in the cytoplasm, and also could be expressed in mice

  18. The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 μmol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The ex- pression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regu- lated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation

  19. 人NESG1基因慢病毒载体的构建及其在293FT细胞中的表达%Construction of a lentiviral vector containing human NESG1 gene and its expression in 293FT cells

    Institute of Scientific and Technical Information of China (English)

    刘真; 甄艳; 于晓黎; 江庆萍; 龙捷; 方唯意

    2011-01-01

    目的 克隆人NESG1基因,构建与增强型绿色荧光蛋白(EGFP)融合表达的慢病毒载体pGC-FU-NESG1-EGFP,包装慢病毒,感染293FT细胞并进行鉴定.方法 以NESG1全长克隆为模板扩增其编码框序列,通过限制性内切酶AgeⅠ酶切、T4DNA连接酶连接,将NESG1插入慢病毒载体pGC-FU-EGFP,构建pGC-FU-NESG1-EGFP重组载体.质粒转化感受态细菌,筛选阳性克隆,经PCR及测序鉴定正确后通过脂质体将慢病毒三质粒系统共转染人胚肾细胞系293FT,进行慢病毒包装并测定病毒滴度.病毒感染293FT细胞,荧光显微镜下观察感染效率,Western blot方法检测NESG1-EGFP融合蛋白的表达情况.结果 成功构建慢病毒表达载体pGC-FU-NESG1-EGFP,三质粒共转染293FT细胞后可见大量绿色荧光;浓缩病毒后测定其滴度为2×107TU/ml;以复感染系数MOI为1感染293FT细胞,感染效率在90%以上.Western blot证实细胞表达NESG1.结论 成功构建NESG1慢病毒表达载体,包装得到高滴度慢病毒,为后续感染鼻咽癌细胞,探索其在鼻咽癌发生和发展中的作用奠定了基础.%Objective To construct a lentiviral vector carrying human NESG1-EGFP gene and observe its expression in 293FT cells. Methods The CDS region of NESG1 gene was amplified from a plasmid containing the full-length NESG1 sequence and cloned into the lentiviral vector pGC-FU-EGFP by restriction endonuclease Agel digestion and T4 DNA ligase ligation. After transformation into competent E. Coli cells, the candidate clones were identified by PCR and sequencing. The recombinant plasmid and the two packaging plasmids were co-transfected into human embryonic kidney cell line 293FT cells by lipofectamine 2000 to produce the lentiviral particles, and the viral titer was determined. The 293FT cells were infected by the lentiviral particles obtained and the transfection efficiency was assessed under fluorescent microscope. Western blotting was used to detect the expression of NESG1

  20. After Effects expressions

    CERN Document Server

    Geduld, Marcus

    2013-01-01

    Put the power of Expressions to work in your animations with controls and efficiencies impossible to achieve with traditional keyframing techniques. No programming skills are required. Foundation concepts and skills orient the new designer and serve as a handy reference to the experienced one. Basics of creating expressions, variables, commands, and expression helpers precede the leap into javascript and math essentials for more advanced expressions that include randomness, physical simularions and 3D. Full color illustrations display the scripts and the resulti

  1. Extrachromosomal inducible expression

    NARCIS (Netherlands)

    Veltman, Douwe M; Van Haastert, Peter J M

    2013-01-01

    Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled exp

  2. The space-specific expression of HSD1 protein during spermatogenesis%HSD1蛋白在精子发生过程中的空间特异性表达

    Institute of Scientific and Technical Information of China (English)

    宋伟; 陈迎春; 缪时英; 王琳芳

    2013-01-01

    Objective To study the space-specific expression and nucleocytoplasmic shuttling of HSD1 in different spermatogenic cells. Methods Mouse spermatogenic cells were isolated and endogenous positioning of HSD1 was observed by immunofluorescence assay. pEGFP-C1-HSD1 expression plasmid was constructed and transfected into CHO cells. Confocal and electron microscopy were used to observe the positioning and nucleocytoplasmic shuttling of HSD1 in cells. Results HSD1 was expressed in the cytoplasm of primary spermatocytes and round spermatids; while it was mainly expressed in the acrosome and tail of mature sperms. There were three distribution forms of HSD1 protein in CHO cells; the nucleus type, the cytoplasm type and the nucleocytoplasmic type. Further study found that distribution of HSD1 was dynamic, and it existed nucleocytoplasmic shuttling in CHO cells. Conclusions HSD1 protein is space-specifically expressed in spermatogenic cells and has the ability of nucleocytoplasmic shuttling.%目的 研究HSD1蛋白在各级生精细胞中的空间特异性表达及其在细胞中的核质穿梭特性.方法 分离小鼠生精细胞,用免疫荧光法观察HSD1蛋白的内源定位.构建pEGFP-C1-HSD1融合表达质粒并转染CHO细胞,用激光共聚焦和电镜观察HSD1融合蛋白的定位及其核质分布情况.结果 HSD1蛋白在初级精母细胞和圆形精子细胞的胞质中表达;在成熟精子细胞中主要表达于顶体和尾部.HSD1蛋白在CHO细胞中存在3种分布形式:细胞核型、细胞质型和核质分布型.进一步研究发现该蛋白在细胞中呈动态分布,存在细胞核-质间的穿梭特性.结论 HSD1蛋白在生精细胞中呈空间特异性表达并且具有核质穿梭能力.

  3. Prokaryotic expression and polyclonal antibody preparation of enterovirus 71 3A protein%人肠道病毒71型3A蛋白的原核表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    黄琴琴; 唐瑞; 杨勇波

    2013-01-01

    Objectives To construct a prokaryotic expression vector of the EV71 3a gene and prepare its recombinant protein and polyclonal antibody for subsequent study.Methods The 3a gene was amplified with PCR and cloned into the vector PET28a to yield PET28a-3a for the prokaryotic expression of 3A protein.The recombinant 3A protein was expressed in E.coli BL21 and was subsequently used to immunize mice.The resulting anti-sera were evaluated using immunofluorescence staining and Western blotting.Results The recombinant protein 3A was efficiently produced in E.coli in the form of inclusion bodies in the expressed protein.Western blot analysis indicated that the resulting mouse anti-3A sera reacted with the eukaryotically expressed EGFP-3A fusion protein.Moreover,the antisera positively recognized cells infected with EV71 according to immunofluorescence staining.Conclusion An anti-3A antibody was successfully prepared and may provide a foundation for subsequent study of the 3a gene.%目的 构建人肠道病毒71型3A基因原核表达质粒,制备重组3A蛋白及其抗体. 方法 PCR方法扩增人肠道病毒71型3A基因,构建原核表达质粒PET28a-3A并转化大肠埃希菌,诱导表达3A重组蛋白,免疫小鼠制备3A蛋白抗体.通过免疫荧光和Western blot方法鉴定抗体特异性. 结果 成功构建了PET28a-3A原核表达质粒并表达了3A重组蛋白,3A蛋白以包涵体的形式存在.细胞免疫荧光和Western blot检测显示,制备的鼠源3A蛋白抗体可识别真核表达的EGFP-3A融合蛋白以及EV71感染细胞表达的3A蛋白. 结论 成功构建了人肠道71型3A基因原核表达质粒,利用该质粒表达的重组蛋白成功制备了特异性的3A蛋白的鼠源抗体.

  4. Construction and stable expression of pLEGFP-N1-stromal cell-derived factor-1α in bone marrow stromal cells from rhesus%pLEGFP-N1-基质细胞衍生因子-1α的构建与目的基因在恒河猴骨髓基质细胞内的稳定表达

    Institute of Scientific and Technical Information of China (English)

    李三清; 徐强; 刘柯; 许百男; 潘力; 饶军华

    2010-01-01

    目的 构建携带人基质细胞衍生因子(SDF)1α基因的反转录病毒真核表达载体pLEGFP-N1-SDF-1α,转染恒河猴骨髓基质细胞(BMSC),观察外源性SDF-1α和增强型绿色荧光蛋白(EGFP)基因在BMSC中的表达情况.方法 应用基因重组技术,从pBudce4.1-SDF-1α获得SDF-1α基因片段,重组到pLEGFP-N1真核表达载体上.pLEGFP-N1-SDF-1α经病毒包装,转染至恒河猴BMSC,用Western免疫印迹和免疫细胞化学检测表达情况.结果 酶切、PCR和DNA序列鉴定均证实插入基因片段的正确性.BMSC转染pLEGFP-N1-SDF-1α后,在荧光显微镜下发出绿色荧光.western免疫印迹和免疫细胞化学证实SDF-1α在细胞内有效表达.结论 成功构建本研究pLEGFP-N1-SDF-1α,经病毒包装转染至恒河猴BMSC,SDF-1α和EGFP基因在BMSC内有效表达.为BMSC-SDF-1α-EGFP工程细胞自体移植治疗相关疾病提供了依据.%Objective To construct a retrovirus eukaryotic expression vector pLEGFP-N1-stromal cell-derived factor-1α (SDF)- 1α that contains human SDF-1α and transfects bone marrow stromal cells (BMSCs) of rhesus, and to examine the expression of exogenous SDF- 1α and EGFP genes in BMSCs .Methods SDF-1α gene obtained from pBudce4.1-SDF-1α was recombined into pLEGFP-N1 vector to generate pLEGFP-N1-SDF-1α by use of genetic recombination techniques. pLEGFP-N1-SDF-1α packaged in virus was transfected into BMSCs of rhesus. Western blotting and immunocytochemistry were used for detection of its expression. Results The inserted gene was verified by enzyme restriction analysis, PCR and DNA sequencing. After transfected with pLEGFP-N1-SDF-1α, the BMSCs emitted green fluorescence, and expressed SDF- 1α as confirmed by Western blotting and immunocytochemistry. Conclusion After transfected to BMSCs of rhesus in virus, pLEGFP-N 1-SDF-1α may effectively express SDF-1α and EGFP,which provides evidences for auto-grafting of BMSC-SDF-1α-EGFP engineered cells in treatment of certain

  5. Expression of Mouse SCP2 Gene Adenoviral Vector Carrying Albumin Promoter in Hepa1-6 Cells%固醇携带蛋白2腺病毒载体的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    贾岩峰; 崔云峰; 崔乃强; 彭雁飞; 宁召臣; 张琚

    2012-01-01

    Objectives To construct the replication defective adenoviral vector of SCP2 gene carrying murine albumin promoter, and study the relations between SCP2 gene and the formation of cholesterol calculus. Methods The cDNA of SCP2 gene was cloned by using RT-PCR technique. The albumin promoter was linked to SCP2 gene's upstream, and the EGFP gene lied in its downstream. The plasmid pDC312-ALB-SCP2-IRES2 -EGFP was constructed by the gene recombination technique. The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors, which were purified by CsCl method. The processes of TCID50 were applied to detect the titers of the adenoviral vectors. The RNA and protein were respectively extracted from the infected Hepal-6 cells by the adenoviral vector. The real-time quantitative PCR was employed to detect the mRNA expression levels, and the Western blotting analysis was used to measure the SCP2 protein levels. Result We constructed successfully the replication defective adenoviral vector of SCP2 gene carrying murine albumin promoter. When the mRNA levels of SCP2 gene were overexpressed, CYP7al mRNA levels were down-regulated (t=3.97,p<0.05); and the mRNA levels of HMGCR were up-regulated (t=3.23,p<0.05). Conclusions The SCP2 gene overexpression may affect cholesterol and bile acid metabolism, which could promote the formation of cholesterol calculus.%目的:构建携带白蛋白启动子SCP2 基因腺病毒载体,研究其与胆固醇结石形成的关系.方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)采用Ad Max TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度;(3)重组腺病毒感染小鼠hepa-1-6细胞,实时定量PCR检测mRNA的表达;Western印迹检测SCP2蛋白表达情况;结果:成功构建携带白蛋白启动子SCP2基因腺病毒载体;当SCP2

  6. 人14-3-3β蛋白的原核表达、抗血清制备及真核表达载体的构建%Prokaryotic expression, antiserum preparation and construction of eukaryotic expression vector of human 14-3-3β protein

    Institute of Scientific and Technical Information of China (English)

    杨学习; 孙敏英; 马瑞娟; 徐伟文; 李明

    2009-01-01

    目的 纯化原核表达的14-3-3β(YWHAB)重组蛋白并制备多抗血清,构建适用于哺乳动物细胞的真核表达载体.方法 将重组蛋白表达载体pET30a(+)/YWHAB转化大肠杆菌表达菌株BL21(DE3)感受态细胞,异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,镍-四齿螯合剂(Ni-NTA)亲和层析柱纯化重组蛋白;以纯化的重组蛋白为抗原免疫BALB/c小鼠,应用ELISA和Western blot方法分别检测抗血清的效价和特异性;应用PCR扩增添加BamH Ⅰ和EcoR Ⅰ酶切位点把YWHAB的ORF亚克隆至真核表达载体pEGFP-N1,添加BamH Ⅰ和Hind Ⅲ酶切位点把YWHAB的开放阅读框(ORF)亚克隆至真核表达载体pCDNA3.1(+),对重组载体进行酶切和PCR鉴定.结果 YwHAB重组蛋白以可溶性形式表达,分子量为32 000,与预期分子量一致;纯化后的重组蛋白纯度达90%以上,ELISA结果显示其抗血清的效价为1:50 000,Western blot结果表明抗血清的特异性较好;酶切和PCR鉴定结果表明真核表达载体pEGFP-N1/YWHAB和pCDNA3.1(+)YWHAB构建成功.结论 通过亲和层析纯化获得人14-3-3β重组蛋白,进而免疫BALB/c小鼠制备多抗血清,为进一步研究人14-3-3β的功能成功构建了其真核表达载体.%Objective To purify human 14-3-3β (YWHAB) recombinant protein expressed in the E.coli, prepare its antiserum and construct the eukaryotic expression vector for transfecting mammalian cells. Methods The human 14-3-313 recombinant protein expression vector pET30a (+) /YWHAB constructed by the ORF of YWHAB gene and prokaryotic expression vector pET30a (+) was transformed into E.coli BL21 (DE3). The expression of the recombinant protein was induced by IPTG and the protein was purified by affinity chromatography on a Ni-NTA resin. BALB/c mice were immunized by the purified protein, and ELISA and Western blotting were employed to detect the titer and specificity of the antiserum. The open reading flame of YWHAB gene was obtained by PCR

  7. 耐辐射球菌pprI基因真核表达载体的构建及其抗辐射作用%Construction of eukaryotic expression vector carrying pprI gene of Deinococcus radiodurans and its radioresistant effect

    Institute of Scientific and Technical Information of China (English)

    文玲; 施怡; 任丽丽; 丛瑛; 杨占山

    2014-01-01

    目的 研究耐辐射球菌pprI原核基因的真核表达载体的构建及其转染对离体真核细胞急性放射损伤的防护作用.方法 应用DNA重组技术构建pEGFP-c1-pprI质粒;采用脂质体转染技术分别将空载体质粒pEGFP-c1和重组质粒pEGFP-c1-pprI转入人肺上皮细胞系Beas-2B细胞,筛选稳定转染细胞系;应用集落形成实验检测受照转染细胞的生存能力;流式细胞仪检测细胞周期和凋亡率的变化;荧光显微镜观察受照细胞ROS荧光强度;免疫荧光实验观察受照细胞不同时间γ-H2AX焦点数目.结果 成功地构建了pprI原核基因的真核表达载体,并在Beas-2B细胞中表达了PprI融合蛋白,建立了稳定转染细胞系.与空白对照组和空载体组相比,细胞克隆生存曲线显示转染了pprI基因的Beas-2B细胞经不同剂量辐射,其存活分数SF增加,该曲线参数D0、Dq、N增高;转染了pprI基因的Beas-2B细胞受照后的ROS的荧光强度和不同时间(1、2、4 h) γ-H2AX的焦点数均显著减低(F=16.73、19.47、6.94,P<0.05);此外,该细胞G2期阻滞(F=139.73、237.92,P<0.05)和凋亡率显著减低(F=626.02、2 052,P<0.05).结论 耐辐射球菌pprI原核基因可在离体真核细胞中稳定表达,并且显著提高受照真核细胞的辐射抗性.%Objective To construct the eukaryotic expression vector of pprI gene from Deinococcus radiodurans R1 and investigate its radioresistant effects in eukaryotic cells.Methods A recombinant vector pEGFP-c1-pprI was constructed by DNA recombinant technique.The empty vector pEGFP-c1 and the pEGFP-c1-pprI were transferred into human lung epithelial cells Beas-2B by LipofectamineTM 2000,respectively.Then the infected cells were screened in order to develop a cell line with stable expression of pprI gene.Cell survival rate was tested by clone-forming assay.Cell cycle distribution and apoptosis were detected by a flow cytometry.The fluorescence intensity of reactive oxygen species (ROS

  8. 外源性分化抑制因子Id2在C2C12细胞中的表达%The expression of external Id2 protein gene containing green fluorescence in C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    赖桂华; 余磊; 张黎声; 欧阳钧; 邱小忠

    2012-01-01

    目的:构建大鼠Id2基因真核荧光表达载体,并观察外源性Id2基因C2C12细胞中的表达.方法:RT-PCR扩增出Id2全长cDNA,T4 DNA连接酶将载体pGEM-T和Id2 cDNA进行连接,构建克隆载体,经限制性内切酶EcoR I酶切pGEM-Id2克隆载体和pEGFP-C2真核表达载体,构建出重组真核表达载体pEGFP-C2-Id2,经酶切分析、PCR鉴定及DNA测序证实cDNA片段大小和序列的正确性;通过电穿孔转染法将外源性Id2基因导入C2C12成肌细胞中.分别于转染4、8、12、24、36、72 h后通过荧光倒置显微镜下观察细胞整体情况,并计算转染效率.结果:经酶切分析和序列测定证实pEGFP-C2-Id2含大小正确的正向Id2 cDNA片段,获得高转染率和高表达外源性Id2基因的C2C12细胞,转染8h时,转染效率约为(10.5±2.8)%;转染12 h后,转染效率约为(20.9±3.1)%;转染24 h后,转染效率最高,约为(60.8±3.2)%.结论:成功构建了同时携带有G418筛选位点和Id2基因的真核表达载体;并获得高表达外源性Id2基因的C2C12细胞.%Objective:To construct the eukaryotic expression vector of rat Id2 and to observe the expression of 1(12 in CZC|2 cells for further study on skeletal muscle regeneration. Methods; RT-PCR method was used to amplify the entire Id2 cDNA. The pGEM-T and Id2 cDNA were ligated by T4 DNA ligase. The cloning vectors and the pEGFP-C2 (eukaryotic expression vector) were first cut by EcoR I and then ligated with Id2 by T4 DNA ligase again. The enzyme analysis and DNA sequencing were used to confirm the recombined vectors. The pEGFP-C2-Id2 vectors were transferred into C2C,2 cells by electric perforation. Fluorescence inverted microscopy was used to observe the global growth of the cells and to calculate the transfection efficiency 4,8,12,24,36 and 72 hours post-transfection. Results:The enzyme analysis and DNA sequencing analysis confirmed that the right Id2 gene was cloned. The Id2 transferred C2C12 cells with high expression and high

  9. Holistic facial expression classification

    Science.gov (United States)

    Ghent, John; McDonald, J.

    2005-06-01

    This paper details a procedure for classifying facial expressions. This is a growing and relatively new type of problem within computer vision. One of the fundamental problems when classifying facial expressions in previous approaches is the lack of a consistent method of measuring expression. This paper solves this problem by the computation of the Facial Expression Shape Model (FESM). This statistical model of facial expression is based on an anatomical analysis of facial expression called the Facial Action Coding System (FACS). We use the term Action Unit (AU) to describe a movement of one or more muscles of the face and all expressions can be described using the AU's described by FACS. The shape model is calculated by marking the face with 122 landmark points. We use Principal Component Analysis (PCA) to analyse how the landmark points move with respect to each other and to lower the dimensionality of the problem. Using the FESM in conjunction with Support Vector Machines (SVM) we classify facial expressions. SVMs are a powerful machine learning technique based on optimisation theory. This project is largely concerned with statistical models, machine learning techniques and psychological tools used in the classification of facial expression. This holistic approach to expression classification provides a means for a level of interaction with a computer that is a significant step forward in human-computer interaction.

  10. RNase E affects the expression of the acyl-homoserine lactone synthase gene sinI in Sinorhizobium meliloti.

    Science.gov (United States)

    Baumgardt, Kathrin; Charoenpanich, Pornsri; McIntosh, Matthew; Schikora, Adam; Stein, Elke; Thalmann, Sebastian; Kogel, Karl-Heinz; Klug, Gabriele; Becker, Anke; Evguenieva-Hackenberg, Elena

    2014-04-01

    Quorum sensing of Sinorhizobium meliloti relies on N-acyl-homoserine lactones (AHLs) as autoinducers. AHL production increases at high population density, and this depends on the AHL synthase SinI and two transcriptional regulators, SinR and ExpR. Our study demonstrates that ectopic expression of the gene rne, coding for RNase E, an endoribonuclease that is probably essential for growth, prevents the accumulation of AHLs at detectable levels. The ectopic rne expression led to a higher level of rne mRNA and a lower level of sinI mRNA independently of the presence of ExpR, the AHL receptor, and AHLs. In line with this, IPTG (isopropyl-β-D-thiogalactopyranoside)-induced overexpression of rne resulted in a shorter half-life of sinI mRNA and a strong reduction of AHL accumulation. Moreover, using translational sinI-egfp fusions, we found that sinI expression is specifically decreased upon induced overexpression of rne, independently of the presence of the global posttranscriptional regulator Hfq. The 28-nucleotide 5' untranslated region (UTR) of sinI mRNA was sufficient for this effect. Random amplification of 5' cDNA ends (5'-RACE) analyses revealed a potential RNase E cleavage site at position +24 between the Shine-Dalgarno site and the translation start site. We postulate therefore that RNase E-dependent degradation of sinI mRNA from the 5' end is one of the steps mediating a high turnover of sinI mRNA, which allows the Sin quorum-sensing system to respond rapidly to changes in transcriptional control of AHL production.

  11. Construction and identification of the PTEN expression plasmid GFP-PTEN%PTEN基因真核表达载体的构建及在MG63中表达

    Institute of Scientific and Technical Information of China (English)

    徐生林; 胡勇; 金问森; 王明明; 王京; 王娟

    2012-01-01

    目的 构建PTEN基因真核表达载体,为PTEN基因功能研究提供工具.方法 从人淋巴细胞中提取总RNA,采用反转录-聚合酶链反应扩增PTEN基因编码区,将其克隆入pEGFP-N1载体中.通过聚合酶链反应、酶切和DNA测序鉴定所构建的载体.脂质体包裹重组载体转染骨肉瘤MG63细胞,RT-PCR和Western blot检测转染后的骨肉瘤MG63细胞中PTEN基因mRNA和蛋白质表达情况.结果 经过限制性酶切和测序鉴定得到重组子GFP-PTEN大小符合,序列与GenBank中人DNA的PTEN基因(NM_000314)完全一致.转染GFP-PTEN基因的骨肉瘤MG63细胞中PTEN基因mRNA和蛋白高水平表达.结论 成功地构建了PTEN基因真核表达载体.%Objective To construct an eukaryotic expression vector for phosphatase and tensin homology deletedon chromosome ten( PTEN ) gene lymphocytes and provide a tool for studying of PTEN gene function. Methods The total RNAs were isolated from human lymphocytes. The cDNA of PTEN gene was amplified by reverse transcription polymerase chain reaction( RT-PCR ). After purification, the gene was cloned into pEGFP-Nl vector. The recombi-nant plasmid was identified by enzyme digestion and DNA sequencing. Then GFP and GFP-PTEN were respectively transfected into the osteosaocoma cell line( MG63 )with Lipofectamine 2000. The mRNA and protein expression level of PTEN gene in each cell group was detected by fluorescent quantitative RT-PCR and Western blot. Results Re-combinant vector of GFP-PTEN was constructed successfully. After transfection with GFP-PTEN, the mRNA and protein expression level of PTEN rose in MG63 cells. There were significant differences between transfected group and control group. Conclusion The eukaryotic expression vector of PTEN gene has been successfully constructed, which may provide a basis for further researches.

  12. Transplantation of D15A-Expressing Glial-Restricted-Precursor-Derived Astrocytes Improves Anatomical and Locomotor Recovery after Spinal Cord Injury

    Directory of Open Access Journals (Sweden)

    Chunling Fan, Yiyan Zheng, Xiaoxin Cheng, Xiangbei Qi, Ping Bu, Xuegang Luo, Dong H. Kim, Qilin Cao

    2013-01-01

    Full Text Available The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI. In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs-derived astrocytes (GDAs could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs. Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.

  13. Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR.

    Science.gov (United States)

    Hu, Bin; Yang, Yan; Liu, Jia; Ma, Zhiyong; Huang, Hongping; Liu, Shenpei; Yu, Yuan; Hao, Youhua; Wang, Baoju; Lu, Mengji; Yang, Dongliang

    2010-10-01

    To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

  14. Potent anti-melanoma effect by combination of mytomycin C with recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO%丝裂霉素C与表达Smac/DIABLO的肿瘤靶向腺相关病毒联用对抗恶性黑色素瘤的效果

    Institute of Scientific and Technical Information of China (English)

    王坚; 林茂; 吴平; 何惠娟; 刘新垣

    2012-01-01

    To investigate whether recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO can improve sensitivity of melanoma to mitomycin C both in vitro and in vivo. Methods Tumor-targeting expressed Smac/DIABLO or green fluorescence protein (EGFP) in recombinant adeno-associated virus vectors (rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP) was constructed. The culture cells were transfected with rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP. The expression of EGFP in culture cells was observed with fluorescence microscope. Smac/DIABLO expression was detected by RT-PCR method. Proliferation of tumor cells was measured by MTT method. Apoptosis of tumor cells at different drug concentrations was examined by flow cytometry. Synergistic anti-tumor activity of mitomycin C combined with rAAV-hTERT/ Smac/DIABLO was measured by MTT in vitro and animal experiment in vivo. Data was evaluated by SPSS statisticssoftware analysis. Results Green fluorescence could be observed in tumor cells but not in normal cells 48 h after rAAV-hTERT/EGFP transfection. Almost all tumor cells displayed bright yellow-green fluorescence after 96 h. The expression of Smac/DIABLO in rAAV-hTERT/Smac/DIABLO transfected tumor cells showed Smac/DIABLO mRNA band 24 h after transfection and stabilized 48 h after transfection. Tumor cell inhibition rate was increased obviously higher in group of combination of mitomycin C with rAAV-hTERT/Smac/DIABLO than mitomycin C alone (P<0.01). Flow cytometry results indicated that mitomycin C combined with rAAV-hTERT/Smac/DIABLO group had the highest apoptosis-induced effect in groups of negative, mitomycin C, and rAAV-hTERT/Smac/ DIABLO (P<0.01). Animal experiment result indicated that tumor growth was inhibited and survival rate was improved significantly in mitomycin C combined with rAAV-hTERT/Smac/DIABLO group compared to rAAV-hTERT/Smac/DIABLO or mitomycin C aione. Conclusion Tumor-targeting rAAV-hTERT/Smac/DIABLO can improve sensitivity of tumor cells

  15. Regular Expression Pocket Reference

    CERN Document Server

    Stubblebine, Tony

    2007-01-01

    This handy little book offers programmers a complete overview of the syntax and semantics of regular expressions that are at the heart of every text-processing application. Ideal as a quick reference, Regular Expression Pocket Reference covers the regular expression APIs for Perl 5.8, Ruby (including some upcoming 1.9 features), Java, PHP, .NET and C#, Python, vi, JavaScript, and the PCRE regular expression libraries. This concise and easy-to-use reference puts a very powerful tool for manipulating text and data right at your fingertips. Composed of a mixture of symbols and text, regular exp

  16. Regular expressions cookbook

    CERN Document Server

    Goyvaerts, Jan

    2009-01-01

    This cookbook provides more than 100 recipes to help you crunch data and manipulate text with regular expressions. Every programmer can find uses for regular expressions, but their power doesn't come worry-free. Even seasoned users often suffer from poor performance, false positives, false negatives, or perplexing bugs. Regular Expressions Cookbook offers step-by-step instructions for some of the most common tasks involving this tool, with recipes for C#, Java, JavaScript, Perl, PHP, Python, Ruby, and VB.NET. With this book, you will: Understand the basics of regular expressions through a

  17. Darwin and Emotion Expression

    Science.gov (United States)

    Hess, Ursula; Thibault, Pascal

    2009-01-01

    In his book "The Expression of the Emotions in Man and Animals," Charles Darwin (1872/1965) defended the argument that emotion expressions are evolved and adaptive (at least at some point in the past) and serve an important communicative function. The ideas he developed in his book had an important impact on the field and spawned rich domains of…

  18. Facial expression and sarcasm.

    Science.gov (United States)

    Rockwell, P

    2001-08-01

    This study examined facial expression in the presentation of sarcasm. 60 responses (sarcastic responses = 30, nonsarcastic responses = 30) from 40 different speakers were coded by two trained coders. Expressions in three facial areas--eyebrow, eyes, and mouth--were evaluated. Only movement in the mouth area significantly differentiated ratings of sarcasm from nonsarcasm.

  19. Long-term reproducible expression in human fetal liver hematopoietic stem cells with a UCOE-based lentiviral vector.

    Directory of Open Access Journals (Sweden)

    Niraja Dighe

    Full Text Available Hematopoietic Stem Cell (HSC targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC. Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001. While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP, only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001. In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg-/- demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.

  20. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

    Directory of Open Access Journals (Sweden)

    Cheng-Tao Yang

    Full Text Available BACKGROUND: The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. METHODOLOGY/PRINCIPAL FINDINGS: To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. CONCLUSIONS/SIGNIFICANCE: These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

  1. EXPRESS Rack Mockup

    Science.gov (United States)

    2002-01-01

    The EXPRESS Rack is a standardized payload rack system that transports, stores, and supports experiments aboard the International Space Station (ISS). EXPRESS stands for EXpedite the PRocessing of Experiments to the Space Station, reflecting the fact that this system was developed specifically to maximize the Station's research capabilities. The EXPRESS Rack system supports science payloads in several disciplines, including biology, chemistry, physics, ecology, and medicine. With the EXPRESS Rack, getting experiments to space has never been easier or more affordable. With its standardized hardware interfaces and streamlined approach, the EXPRESS Rack enables quick, simple integration of multiple payloads aboard the ISS. The system is comprised of elements that remain on the ISS, as well as elements that travel back and forth between the ISS and Earth via the Space Shuttle. The Racks stay on orbit continually, while experiments are exchanged in and out of the EXPRESS Racks as needed, remaining on the ISS for three months to several years, depending on the experiment's time requirements. A refrigerator-sized Rack can be divided into segments, as large as half of an entire rack or as small as a bread box. Payloads within EXPRESS Racks can operate independently of each other, allowing for differences in temperature, power levels, and schedules. Experiments contained within EXPRESS Racks may be controlled by the ISS crew or remotely by the Payload Rack Officer at the Payload Operations Center at the Marshall Space Flight Center (MSFC). The EXPRESS Rack system was developed by MSFC and built by the Boeing Co. in Huntsville, Alabama. Eight EXPRESS Racks are being built for use on the ISS.

  2. Regulation of melanopsin expression.

    Science.gov (United States)

    Hannibal, Jens

    2006-01-01

    Circadian rhythms in mammals are adjusted daily to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Retinal ganglion cells (RGCs) of the RHT constitute a separate light-detecting system in the mammalian retina used for irradiance detection and for transmission to the circadian system and other non-imaging forming processes in the brain. The RGCs of the RHT are intrinsically photosensitive due to the expression of melanopsin, an opsin-like photopigment. This notion is based on anatomical and functional data and on studies of mice lacking melanopsin. Furthermore, heterologous expression of melanopsin in non-neuronal mammalian cell lines was found sufficient to render these cells photosensitive. Even though solid evidence regarding the function of melanopsin exists, little is known about the regulation of melanopsin gene expression. Studies in albino Wistar rats showed that the expression of melanopsin is diurnal at both the mRNA and protein levels. The diurnal changes in melanopsin expression seem, however, to be overridden by prolonged exposure to light or darkness. Significant increase in melanopsin expression was observed from the first day in constant darkness and the expression continued to increase during prolonged exposure in constant darkness. Prolonged exposure to constant light, on the other hand, decreased melanopsin expression to an almost undetectable level after 5 days of constant light. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 days. These observations show that dual mechanisms regulate melanopsin gene expression and that the intrinsic light-responsive RGCs in the albino Wistar rat adapt their expression of melanopsin to environmental light and darkness.

  3. Study on Up-Regulated Expressions of Anti-HIV Genes by vMIP-Ⅰin Jurkat Cell%vMIP-Ⅰ激活Jurkat细胞抗-HIV基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    尹小菲; 陈彬; 谭晓华; 罗燕; 杨磊

    2012-01-01

    本研究通过构建真核表达载体pEGFP-N3-vMIP-Ⅰ,电穿孔法将其转染至Jurkat细胞,荧光定量PCR检测vMIP-Ⅰ基因对Jurkat细胞内CCL5、APOBEC3G、APOBEC3F、等抗-HIV基因表达水平的影响,从而探讨vMIP-Ⅰ抗HIV感染的机制.结果显示:成功构建了pEGFP-N3-vMIP-Ⅰ载体,电穿孔转染效率达到40%左右,与转染空载体组相比,vMIP-Ⅰ转染组的Jurkat细胞内CCL5、A3G、A3F和MX1分别上调7.37倍、1.58倍、2.42倍和2.06倍.研究结果表明:vMIP-Ⅰ基因可激活Jurkat细胞内一些抗HIV相关基因的表达,这可能是vMIP-Ⅰ基因抗HIV感染的机制之一.%In this study, through the construction of eukaryotic expressive vector, the recombinant plasmids Pegfp-N3-Vmip- 玉 was transfected into Jurkat cells by electroporation. Then, by QRT-PCR technique, we detected the expression levels of anti-HIV genes: CCL5, APOBEC3F, MX1 in Jurkat cells which influenced by Vmip- 玉 gene and explored the mechanisms of Vmip- 玉 against HIV infection. The results he recombinant plasmids of Pegfp-N3-Vmip- 玉 was successfully constructed and electroporation transfection efficiency reached about 40%. In comparison with non-transfected gourp, the transfected Vmip- 玉 group can increase CCL5, A3G, A3F, and MX1 of Jurkat cells by 7.37, 1.58, 2.42 and 2.064 times seperately. The results suggest Vmip- 玉 can activate expressions of some relative anti-HIV genes in Jurkat cells, which probably is one of the mechanisms for anti-HIV infection.

  4. 嗜肺军团菌mip基因重组质粒GFP-mip的构建及表达%The Construction and Expression of Recombinant Plasmid GFP -mip of Legionella Pneumophila Macrophage Infectivity Potentiator Gene

    Institute of Scientific and Technical Information of China (English)

    惠英华; 曹秀琴; 杨志伟

    2011-01-01

    Objective To construct recombinant plasmid GFP - mip of Legionella pneumophila macrophage infectivity potentiator gene and observe its expression in the NIH3T3 cells. Methods The macrophage infectivity potentiator gene was amplified from DNA of Legionella pneumophila by polymerase chain reation ( PCR),then cloned into pEGFP - C1 vector. The recombinant plasmid was named as GFP - mip and was analyzed with restriction endonuclease XhoI and BarnHl digestion, PCR and DNA sequencing techniques. The NIH3T3 cell was transfected by recombinant plasmid GFP - mip with lipofection strategy. The stable expression products of macrophage infectivity petentiator gene were observed by the fluorescent microscope. Results 702bp mip gene was amplified . Under the fluorescent microscope, green fluorescent was observed in the cell cytoplasm and on the cell membrane. Conclusion The recombinant plasmid GFP - mip was constructed successfully and expressed in the NIH3T3 cells.%目的构建嗜肺军团菌mip基因的真核重组质粒GFP-mip,并观察其在NIH3T3细胞中的表达.方法 以嗜肺军团菌DNA为模版,通过PCR扩增获得mip基因,将其定向克隆到绿色荧光质粒pEGFP-C1中,构建真核重组质粒GFP-mip.经限制性核酸内切酶XhoI和BamHI酶切鉴定、PCR和核酸序列分析后,通过脂质体法转染到NIH3T3细胞中,利用荧光显微镜观察重组质粒的稳定表达.结果 扩增出了702bpmip基因,在细胞质和细胞膜观察到较强绿色荧光.结论 成功构建了真核重组质粒GFP-mip,并在NIH3T3细胞中得到了表达.

  5. A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression.

    Directory of Open Access Journals (Sweden)

    Jessica A Hill

    Full Text Available Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements.

  6. Defining Optimized Properties of Modified mRNA to Enhance Virus- and DNA- Independent Protein Expression in Adult Stem Cells and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Frauke Hausburg

    2015-02-01

    Full Text Available Background: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. Methods: Relying on an initially