Sample records for mice expressing egfp

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

    Shima Kentaro


    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.

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

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


    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.

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

    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.

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

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


    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.

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

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


    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...

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

    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(+.

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

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


    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.

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

    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.

  9. 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

    张平; 赵钢勇; 陈凯; 宋月平; 康增军; 苏立凯


    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.

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

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


    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...

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

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


    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.

  12. Effects of clinorotation on COL1A1- EGFP gene expression

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


    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.

  13. 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

    熊敏; 于倩; 崔爱娜; 张力; 朱桂金


    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.

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

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


    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.

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

    Roozbeh Ghodratnama


    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.

  16. 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

    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


    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

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

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


    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.

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

    仲照东; 邹萍; 黄士昂; 胡中波; 刘凌波; 卢运萍


    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.

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

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


    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

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

    Anna Lenice Ribeiro Xavier


    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.

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

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


    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.

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

    Veenhuis Marten


    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

  3. Generation of Recombinant Rabies Virus CVS-11 Expressing eGFP Applied to the Rapid Virus Neutralization Test

    Xianghong Xue


    Full Text Available The determination of levels of rabies virus-neutralizing antibody (VNA provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN using a challenge virus standard (CVS-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT. Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

  4. 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

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


    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

  5. 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

    孙欣; 曾荣; 郭伟韬; 肖启贤; 王斌; 黄云; 林颢


    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.

  6. 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

    梁兵; 袁芳; 殷建瑞; 谭丽华; 高庆春; 高聪


    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

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

    Su Li; Chen Yunzhen; Zhang Xiaogang; She Qiang


    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.

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

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


    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.

  9. 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

    邱明链; 方航荣; 陈丽红; 刘景丰


    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

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

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


    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

  11. Endogenously EGFP-Labeled Mouse Embryonic Stem Cells.

    Zhang, Junli; Rao, Rammohan V; Spilman, Patricia; Mangada, Julie; Xie, Lin; Vitelli, Cathy; Gorostiza, Olivia F; Madden, David T; Zeng, Xianmin; Jin, Kunlin; Hart, Matthew J; Bredesen, Dale E; Galvan, Veronica


    Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping β-actin promoter The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759-769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies.

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

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


    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.

  13. Analysis of the effects of blue light on morphofunctional status of in vitro cultured blastocysts from mice carrying gene of enhanced green fluorescent protein (EGFP).

    Sakharova, N Yu; Mezhevikina, L M; Smirnov, A A; Vikhlyantseva, E F


    We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos.

  14. 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

    于淑珍; 冯冲; 石宁宁; 宋小凤; 潘登科


    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.

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

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


    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.

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

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


    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.

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

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


    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.

  18. Neuroglobin over expressing mice

    Raida, Zindy; Hundahl, Christian Ansgar; Nyengaard, Jens R


    BACKGROUND: Stroke is a major cause of death and severe disability, but effective treatments are limited. Neuroglobin, a neuronal heme-globin, has been advocated as a novel pharmacological target in combating stroke and neurodegenerative disorders based on cytoprotective properties. Using...... thoroughly validated antibodies and oligos, we give a detailed brain anatomical characterization of transgenic mice over expressing Neuroglobin. Moreover, using permanent middle artery occlusion the effect of elevated levels of Neuroglobin on ischemic damage was studied. Lastly, the impact of mouse strain...... genetic background on ischemic damage was investigated. PRINCIPAL FINDINGS: A four to five fold increase in Neuroglobin mRNA and protein expression was seen in the brain of transgenic mice. A β-actin promoter was used to drive Neuroglobin over expression, but immunohistochemistry and in situ hybridization...

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

    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

  20. 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

    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


    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.

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

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


    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

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

    季爱加; 宁喜斌


    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

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

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


    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

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

    Wang Y


    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

  5. 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

    赵爱春; 夏庆友; 鲁成; 向仲怀; 张袁松; 中垣雅雄


    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

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

    Palm Kaia


    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.

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

    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.

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

    Netter, S; Faucheux, M; Théodore, L


    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.

  9. 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.

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


    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.

  10. Production of porcine blastocysts expressed EGFP by handmade cloning%利用改进的手工克隆技术生产转GFP基因猪克隆胚胎

    张鹏; 杨珍珍; 窦红伟; 李伟杭; 律波; Bolund Lars; 杜玉涛; 谭萍萍; 马润林


    Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide.However, the application of SCNT is impeded by overall high costs and low efficiency.Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT.Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP.Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation.To demonstrate the concept, we collected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells.In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%.Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly.Furthermore, the modified method no longer relies on expensive instrument like micromanipulator,facilitating the industrialization of transgenic animal production.%通过体细胞核移植(Somatic cell nuclear transfer,SCNT)培育转基因动物新个体是当前被广泛使用的技术之一,但其生产成本高和转基因囊胚形成率低在很大程度上制约了该技术的应用.文章报告对该技术的一些改进以提高其成功率并降低成本.首先将增强型绿色荧光基因(EGFP)导入猪胎儿成纤维细胞中,通过荧光观察EGFP的表达来筛选适合做细胞核移植的体细胞.这样避免了外源EGFP基因虽已整合至猪基因组但不表达的情况,保证供体细胞100%是表达目标蛋白(绿色荧光蛋白)的细胞;然后利用新一代体细

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

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


    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.

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

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


    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.

  13. 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

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


    -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.

  14. 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

    李杰; 任宏伟; 黄洁虹; 俞梅敏; 茹炳根


    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.

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

    Kofuji Takefumi


    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

  16. 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

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


    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.

  17. 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

    吕梁; 霍继荣; 刘斌; 刘佳; 武捷; 王捷


    [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.

  18. 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

    董训忠; 李建生; 许戈良; 荚卫东; 陈浩; 任维华


    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.

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

    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


    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.

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

    Wang Yi


    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

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

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


    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.

  2. 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

    段彪; 杜海燕; 张荣


    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

  3. The dark side of EGFP: defective polyubiquitination.

    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.

  4. 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

    吕梁; 霍继荣; 刘佳; 张宏斌; 武捷; 王捷


    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

  5. Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity calcium Ruby and its dextran conjugate.

    Luccardini, Camilla; Yakovlev, Aleksey V; Pasche, Mathias; Gaillard, Stéphane; Li, Dongdong; Rousseau, France; Ly, Romain; Becherer, Ute; Mallet, Jean-Maurice; Feltz, Anne; Oheim, Martin


    The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.

  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

    高峰; 陈亚军; 杨学文


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

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


    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.

  8. 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

    梅文瀚; 吴兆平; 徐荣婷; 钱关祥; 卢健


    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.

  9. Cardiac hypertrophy in mice expressing unphosphorylatable phospholemman.

    Boguslavskyi, Andrii; Pavlovic, Davor; Aughton, Karen; Clark, James E; Howie, Jacqueline; Fuller, William; Shattock, Michael J


    Elevation of intracellular Na in the failing myocardium contributes to contractile dysfunction, the negative force-frequency relationship, and arrhythmias. Although phospholemman (PLM) is recognized to form the link between signalling pathways and Na/K pump activity, the possibility that defects in its regulation contribute to elevation of intracellular Na has not been investigated. Our aim was to test the hypothesis that the prevention of PLM phosphorylation in a PLM(3SA) knock-in mouse (in which PLM has been rendered unphosphorylatable) will exacerbate cardiac hypertrophy and cellular Na overload. Testing this hypothesis should determine whether changes in PLM phosphorylation are simply bystander effects or are causally involved in disease progression. In wild-type (WT) mice, aortic constriction resulted in hypophosphorylation of PLM with no change in Na/K pump expression. This under-phosphorylation of PLM occurred at 3 days post-banding and was associated with a progressive decline in Na/K pump current and elevation of [Na]i. Echocardiography, morphometry, and pressure-volume (PV) catheterization confirmed remodelling, dilation, and contractile dysfunction, respectively. In PLM(3SA) mice, expression of Na/K ATPase was increased and PLM decreased such that net Na/K pump current under quiescent conditions was unchanged (cf. WT myocytes); [Na(+)]i was increased and forward-mode Na/Ca exchanger was reduced in paced PLM(3SA) myocytes. Cardiac hypertrophy and Na/K pump inhibition were significantly exacerbated in banded PLM(3SA) mice compared with banded WT. Decreased phosphorylation of PLM reduces Na/K pump activity and exacerbates Na overload, contractile dysfunction, and adverse remodelling following aortic constriction in mice. This suggests a novel therapeutic target for the treatment of heart failure. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.

  10. Post-transcriptional regulation of dopamine D1 receptor expression in caudate-putamen of cocaine-sensitized mice.

    Tobón, Krishna E; Catuzzi, Jennifer E; Cote, Samantha R; Sonaike, Adenike; Kuzhikandathil, Eldo V


    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.

  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

    李劲; 刘琳


    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. A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells.

    Misra, Santosh K; Naz, Sarwat; Kondaiah, Paturu; Bhattacharya, Santanu


    The p53 protein mediated anti-tumor strategy is limited due to the lack of suitable delivery agent with insignificant immunogenic response, serum compatibility, and early and easy detection of the transfected cell population. To overcome these problems, we generated a p53-EGFP-C3 fusion construct which expressed easily detectable green fluorescence protein (GFP) and allowed an estimation of p53 mediated anti-tumor activity. A mixture of cationic cholesterol gemini (Chol-5L) with natural lipid, DOPE (molar ratio 1:4), acronymed as Chol-5LD, formed a nano-liposome as characterized by various physical methods. The prepared clone was evaluated for the expression of GFP and functional p53 in HeLa and two additional cell lines with varied p53 status namely, H1299 (p53(-/-)) and HEK293T (p53(+/+)). Transfected cells were screened using RT-PCR, Western blotting, FACS analysis, MTT, Trypan blue assay and visualized under a fluorescence microscope. The p53-EGFP-C3 fusion protein induced apoptosis in cancer cells as evident from DNA fragmentation, cell cycle analysis, Annexin-V staining and PARP cleavage assays. The transfection and apoptosis induction efficiency of Chol-5LD was significantly higher than commercial reagents Lipofectamine2000 and Effectene irrespective of the cell lines examined. Further it significantly decreases the xenograft tumor volume in nude mice tumors via apoptosis as observed in H&E staining. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    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


    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.

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

    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: [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)


    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.

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

    Xiu-Qun Zhang


    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.

  16. 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

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


    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.

  17. 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

    王玉明; 王静萱


    [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

  18. Htr2a gene and 5-HT2A receptor expression in the cerebral cortex studied using genetically modified mice

    Rodrigo Andrade


    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.

  19. Striatal Neurons Expressing D1 and D2 Receptors are Morphologically Distinct and Differently Affected by Dopamine Denervation in Mice.

    Gagnon, D; Petryszyn, S; Sanchez, M G; Bories, C; Beaulieu, J M; De Koninck, Y; Parent, A; Parent, M


    The loss of nigrostriatal dopamine neurons in Parkinson's disease induces a reduction in the number of dendritic spines on medium spiny neurons (MSNs) of the striatum expressing D1 or D2 dopamine receptor. Consequences on MSNs expressing both receptors (D1/D2 MSNs) are currently unknown. We looked for changes induced by dopamine denervation in the density, regional distribution and morphological features of D1/D2 MSNs, by comparing 6-OHDA-lesioned double BAC transgenic mice (Drd1a-tdTomato/Drd2-EGFP) to sham-lesioned animals. D1/D2 MSNs are uniformly distributed throughout the dorsal striatum (1.9% of MSNs). In contrast, they are heterogeneously distributed and more numerous in the ventral striatum (14.6% in the shell and 7.3% in the core). Compared to D1 and D2 MSNs, D1/D2 MSNs are endowed with a smaller cell body and a less profusely arborized dendritic tree with less dendritic spines. The dendritic spine density of D1/D2 MSNs, but also of D1 and D2 MSNs, is significantly reduced in 6-OHDA-lesioned mice. In contrast to D1 and D2 MSNs, the extent of dendritic arborization of D1/D2 MSNs appears unaltered in 6-OHDA-lesioned mice. Our data indicate that D1/D2 MSNs in the mouse striatum form a distinct neuronal population that is affected differently by dopamine deafferentation that characterizes Parkinson's disease.

  20. Dental enamel structure is altered by expression of dominant negative RhoA in ameloblasts.

    Li, Yong; Pugach, Megan K; Kuehl, Melissa A; Peng, Li; Bouchard, Jessica; Hwang, Soon Y; Gibson, Carolyn W


    Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoA(DN)) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoA(DN) fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoA(DN)-8), intermediate (TgEGFP-RhoA(DN)-2), or high (TgEGFP-RhoA(DN)-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoA(DN) expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride. Copyright © 2011 S. Karger AG, Basel.

  1. [Generation of transgenic mice expressing human lysozyme in mammary gland].

    Yan, Hua; Li, Guo-cai; Sun, Huai-chang


    To evaluate the feasibility of generating animal mammary gland bioreactors expressing human lysozyme (hLYZ). The recombinant vector p205C3-hLYZ, as a result of connecting the hLYZ cDNA with the mammry gland expression vector p205C3, was used to generate transfer genic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 females and 5 males) and 4 (1 females and 3 males) were found to contain the transfer-gene by PCR and Southern blotting respectively. The results of Western blotting indicated that the expressed protein had the same molecular weight as that of normal hLYZ. From the F1 generation on, the mice mated only with their brothers or sisters and a colony of F7 transgenic mice was obtained. Among the offspring, the female transgenic mice maintained and expressed the transfer-gene stably with an expression level as high as 750 mg/L. The expressed protein had strong tissue specificity, and in addition to the mammary glands, some degree of ectropic expression in the spleens and intestines of the transgenic mice was confirmed by dot blotting assay. These data indicate that the mice mammary gland bioreactors expressing hLYZ have been successfully generated.

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

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


    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.

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

    Kawakami Minoru


    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.

  4. AGEMAP: a gene expression database for aging in mice.

    Jacob M Zahn


    Full Text Available We present the AGEMAP (Atlas of Gene Expression in Mouse Aging Project gene expression database, which is a resource that catalogs changes in gene expression as a function of age in mice. The AGEMAP database includes expression changes for 8,932 genes in 16 tissues as a function of age. We found great heterogeneity in the amount of transcriptional changes with age in different tissues. Some tissues displayed large transcriptional differences in old mice, suggesting that these tissues may contribute strongly to organismal decline. Other tissues showed few or no changes in expression with age, indicating strong levels of homeostasis throughout life. Based on the pattern of age-related transcriptional changes, we found that tissues could be classified into one of three aging processes: (1 a pattern common to neural tissues, (2 a pattern for vascular tissues, and (3 a pattern for steroid-responsive tissues. We observed that different tissues age in a coordinated fashion in individual mice, such that certain mice exhibit rapid aging, whereas others exhibit slow aging for multiple tissues. Finally, we compared the transcriptional profiles for aging in mice to those from humans, flies, and worms. We found that genes involved in the electron transport chain show common age regulation in all four species, indicating that these genes may be exceptionally good markers of aging. However, we saw no overall correlation of age regulation between mice and humans, suggesting that aging processes in mice and humans may be fundamentally different.

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

    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.

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

    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


    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.

  7. 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

    郭瑞娟; 李淑红; 张西臣; 李建华; 宫鹏涛; 杨举; 张国才


    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.

  8. Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

    Yun Seong-Jo


    Full Text Available Abstract Background Dimeric human erythropoietin (dHuEPO peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg mice expressing dHuEPO and to investigate the characteristics of these mice. Methods A dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile, was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice. Results A high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47 of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11. We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764. Reverse transcriptase-polymerase chain reaction (RT-PCR and quantitative real-time PCR (qRT-PCR were used for validating the results of the microarray

  9. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal

    Macmaster Suzanne


    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

  10. 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

    陈思思; 王家宁; 黄永章; 郭凌郧; 郑飞


    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.

  11. Expression of interferon inducible protein-10 in pancreas of mice

    Dong Li; Su-Wen Zhu; Dong-Juan Liu; Guo-Liang Liu


    AIM: To investigate the expression of interferon inducible protein-10 (IP-10) in pancreas of mice and to discuss its possible role in the pathogenesis of type 1 diabetes.METHODS: Non-obese diabetic (NOD) mice were used as experiment group and BALB/c mice as non-diabetic prone model. Immunohistochemistry method was used to evaluate the expression of IP-10 in the pancreas of NOD mice and BALB/c mice. Immunoelectron microscope was used to show the location of IP-10 in pancreatic islet β cells.RESULTS: Pancreatic islets were positively stained in all the NOD mice. Insulitis could be found in mice at the age of 4 wk. The weakly positive results were found in control group with no insulitis. Immunoelectron microscopy further demonstrated that IP-10 was produced by pancreatic β cells and stored in cytoplasm of the cells.CONCLUSION: IP-10 can be largely produced in pancreatic islets of NOD mice at the age of 2 wk when there is no significant insulitis, and may play an important part in the pathogenesis of type 1 diabetes by attracting immune cells to infiltrate the pancreatic islets.

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

    Moen Ingrid


    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

  13. Expression of estrogen receptor alpha in preimplantation mice embryos


    Objective:To study the expression of estrogen receptor alpha (ERα) in preimplantation mice embryos.Methods:Mice zygotes were collected from superovulated Kunming mice and cultured in vitro.Embryos at different developmental stages were collected at 0,24,36,48,72 and 96hours after cultivation.The expression of ERα in early mice embryos was detected by reverse transcription-PCR (RT-PCR) and immunocytochemistry.Results:The expression of ERα mRNA was detected in all of the examined embryonic stages.The relative amount of ERα mRNA showed no significant difference between 1-cell stage embryos and 4-cell stage embryos (P>0.05).However,the relative level of ERα mRNA significantly decreased (P<0.05) at 2-cell stage and was the lowest at this stage.Over 2-cell stage,the ERα mRNA relative level would increase and achieve the peak level at blastocyst stage.The location of immunocytochemistry showed that ERα immunopositive cells could be firstly detected at 8-cell stage,after which they are consistently detected until blastocyst stage.In addition,the intensity of ERα positive staining was higher at blastocyst stage compared with that at 8-cell stage and morula stage.Conclusion:ERα is expressed in preimplantation mice embryos in a temporal and spatial pattern and may be involved in regulating the development of early mice embryos,which probably plays crucial roles in early embryonic development.

  14. 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

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


      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

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

    Yanping eZhang


    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.

  16. Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

    Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.


    Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

  17. The effect of AD-VEGF-siRNA on the expression of vascular endothelial growth factor in osteosarcoma-bearing nude mice

    Huaming Xue; Jiong Mei; Yihui Tu; Xuansong Cai; Guang Ojan; Mu Hu


    Objective: To explore the influence of AD-VEGF-siRNA on the expression of vascular endothelial growth factor (VEGF) in neoplasm and blood serum.Methods: Transplantable model of human osteosarcoma was successfully established by the way of subcutaneous injection of VEGF highly expressed human MG63 osteosarcoma cells.These mice were divided randomly into three groups: AD-VEGF-siRNA group, 15 mice; AD-EGFP group, 15 mice; PBS group, 15 mice.Three mice were additionally raised without any treatment.The drug was injected intratumorally 200 IJL at each time, once a day.The total dose of virus was 2×109 pfu.Three osteosarcoma-bearing mice of each group were sacrificed at 11th, 14th ,17th day after the implantation of MG63 cells.The expression of VEGF in implanted tumors and blood serum was detected by ELISA methods.Then the left mice were all sacrificed at the end of experiment (19th day).The expression of VEGF in implanted tumors was detected by RT-PCR and immune histochemistry methods, and that in implanted tumors and blood serum was detected by ELISA methods.Results: (1) Tumors in mice could be seen at 5th day from the implantation of MG63 ceils.(2)The expression of VEGF could be detected in all groups by RT-PCR and immune histochemistry, Which was much lower in the group receiving AD-VEGF-siRNA therapy than two control groups (P<0.05).(3) The expression of VEGF in blood serum of osteosarcoma-bearing mice was much higher than that of three healthy mice by ELISA (P<0.05).(4) The expression of VEGF in blood serum and neoplasm in AD-VEGF-siRNA group was much lower than that in two control groups (P<0.05).Conclusion: AD-VEGF-siRNA could effectively inhibited VEGF expression in vivo.This technology would bring some good references for our therapy of antiangiogenesis in osteosarcoma.

  18. Profiling helper T cell subset gene expression in deer mice

    Hjelle Brian


    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

  19. Encapsulation into complex coacervate core micelles promotes EGFP dimerization

    Nolles, A.; Dongen, Van N.J.E.; Westphal, A.H.; Visser, A.J.W.G.; Kleijn, J.M.; Berkel, Van W.J.H.; Borst, J.W.


    Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer

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

    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: [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)


    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.

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

    Zhilan Chen; Meiling Lu; Dandan Zou; Hailin Wang


    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.

  2. Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice

    Sara X. Chen


    Fluorescent proteins (FPs have great utility in identifying specific cell populations and in studying cellular dynamics in the mouse. To quantify the factors that determine both the expression and relative brightness of FPs in mouse embryonic stem cells (mESCs and in mice, we generated eight different FP-expressing ROSA26 alleles using recombinase-mediated cassette exchange (RMCE. These alleles enabled us to analyze the effects on FP expression of a translational enhancer and different 3′-intronic and/or polyadenylation sequences, as well as the relative brightness of five different FPs, without the confounding position and copy number effects that are typically associated with randomly inserted transgenes. We found that the expression of a given FP can vary threefold or more depending on the genetic features present in the allele. The optimal FP expression cassette contained both a translational enhancer sequence in the 5′-untranslated region (UTR and an intron-containing rabbit β-globin sequence within the 3′-UTR. The relative expressed brightness of individual FPs varied up to tenfold. Of the five different monomeric FPs tested, Citrine (YFP was the brightest, followed by Apple, eGFP, Cerulean (CFP and Cherry. Generation of a line of Cherry-expressing mice showed that there was a 30-fold variation of Cherry expression among different tissues and that there was a punctate expression pattern within cells of all tissues examined. This study should help investigators make better-informed design choices when expressing FPs in mESCs and mice.

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


    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.

  4. Development of Neutralization Assay Using an eGFP Chikungunya Virus

    Cheng-Lin Deng


    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.

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

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


    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.

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

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

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


    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

  8. Expression Analysis of CB2-GFP BAC Transgenic Mice.

    Schmöle, Anne-Caroline; Lundt, Ramona; Gennequin, Benjamin; Schrage, Hanna; Beins, Eva; Krämer, Alexandra; Zimmer, Till; Limmer, Andreas; Zimmer, Andreas; Otte, David-Marian


    The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

  9. Expression Analysis of CB2-GFP BAC Transgenic Mice.

    Anne-Caroline Schmöle

    Full Text Available The endocannabinoid system (ECS is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2. As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

  10. c-Fos expression during temporal order judgment in mice.

    Makoto Wada

    Full Text Available The neuronal mechanisms for ordering sensory signals in time still need to be clarified despite a long history of research. To address this issue, we recently developed a behavioral task of temporal order judgment in mice. In the present study, we examined the expression of c-Fos, a marker of neural activation, in mice just after they carried out the temporal order judgment task. The expression of c-Fos was examined in C57BL/6N mice (male, n = 5 that were trained to judge the order of two air-puff stimuli delivered bilaterally to the right and left whiskers with stimulation intervals of 50-750 ms. The mice were rewarded with a food pellet when they responded by orienting their head toward the first stimulus (n = 2 or toward the second stimulus (n = 3 after a visual "go" signal. c-Fos-stained cell densities of these mice (test group were compared with those of two control groups in coronal brain sections prepared at bregma -2, -1, 0, +1, and +2 mm by applying statistical parametric mapping to the c-Fos immuno-stained sections. The expression of c-Fos was significantly higher in the test group than in the other groups in the bilateral barrel fields of the primary somatosensory cortex, the left secondary somatosensory cortex, the dorsal part of the right secondary auditory cortex. Laminar analyses in the primary somatosensory cortex revealed that c-Fos expression in the test group was most evident in layers II and III, where callosal fibers project. The results suggest that temporal order judgment involves processing bilateral somatosensory signals through the supragranular layers of the primary sensory cortex and in the multimodal sensory areas, including marginal zone between the primary somatosensory cortex and the secondary sensory cortex.

  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

    张敏; 赵文晶; 冯海凉; 刘艳艳; 李占稳; 刘玉琴


    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.

    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. A protocol for generation of transgenic mice by manipulating spermatogonial stem cells in vivo.



    Authors: Lalit Sehgal, Rahul Thorat, Nileema Khapare, Amitabha Mukhopadhaya, Mugdha Sawant & Sorab Dalal ### Abstract This protocol describes a technique for the generation of transgenic mice by in-vivo manipulation of spermatogonial stem cells (SSCs) with a high rate of success. In this study SSCs in pre-pubescent animals were infected in vivo with recombinant lentiviruses expressing EGFP-f and mated with normal females. All male pre-founder mice produced transgenic pups with an ...

  14. 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

    仲念念; 朱伶俐; 王旋; 房娜


    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

  15. Neuropathology in mice expressing mouse alpha-synuclein.

    Claus Rieker

    Full Text Available α-Synuclein (αSN in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN, which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occurred in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are

  16. Unresolved questions from the analysis of mice lacking MCU expression.

    Murphy, Elizabeth; Pan, Xin; Nguyen, Tiffany; Liu, Jie; Holmström, Kira M; Finkel, Toren


    Entry of mitochondrial calcium is believed to play an essential role in regulating bioenergetics and initiating cell death pathways. We have recently described a mouse model lacking MCU expression. Surprisingly, these mice are viable and the cells and tissues from these animals do not exhibit any marked protection from cell death. Here, we discuss our findings as well as potential explanations for some of the more unexpected results.

  17. Age-related vascular gene expression profiling in mice.

    Rammos, Christos; Hendgen-Cotta, Ulrike B; Deenen, Rene; Pohl, Julia; Stock, Pia; Hinzmann, Christian; Kelm, Malte; Rassaf, Tienush


    Increasing age involves a number of detrimental changes in the cardiovascular system and particularly on the large arteries. It deteriorates vascular integrity and leads to increased vascular stiffness entailing hypertension with increased cardiovascular morbidity and mortality. The consequences of continuous oxidative stress and damages to biomolecules include altered gene expression, genomic instability, mutations, loss of cell division and cellular responses to increased stress. Many studies have been performed in aged C57BL/6 mice; however, analyses of the age-related changes that occur at a gene expression level and transcriptional profile in vascular tissue have not been elucidated in depth. To determine the changes of the vascular transcriptome, we conducted gene expression microarray experiments on aortas of adult and old mice, in which age-related vascular dysfunction was confirmed by increased stiffness and associated systolic hypertension. Our results highlight differentially expressed genes overrepresented in Gene Ontology categories. Molecular interaction and reaction pathways involved in vascular functions and disease, within the transforming growth factor-beta (TGF-β) pathway, the renin-angiotensin system and the detoxification systems are displayed. Our results provide insight to an altered gene expression profile related to age, thus offering useful clues to counteract or prevent vascular aging and its detrimental consequences. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

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


    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

  19. Muscle Structure Influences Utrophin Expression in mdx Mice

    Banks, Glen B.; Combs, Ariana C.; Odom, Guy L.; Bloch, Robert J.; Chamberlain, Jeffrey S.


    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin−/− mice. An ∼2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin−/−, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD. PMID:24922526

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

    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


    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.

  1. Kidney EPO expression during chronic hypoxia in aged mice.

    Benderro, Girriso F; LaManna, Joseph C


    In order to maintain normal cellular function, mammalian tissue oxygen concentrations must be tightly regulated within a narrow physiological range. The hormone erythropoietin (EPO) is essential for maintenance of tissue oxygen supply by stimulating red blood cell production and promoting their survival. In this study we compared the effects of 290 Torr atmospheric pressure on the kidney EPO protein levels in young (4-month-old) and aged (24-month-old) C57BL/6 mice. The mice were sacrificed after being anesthetized, and kidney samples were collected and processed by Western blot analysis. Relatively low basal expression of EPO during normoxia in young mice showed significant upregulation in hypoxia and stayed upregulated throughout the hypoxic period (threefold compared to normoxic control), showing a slight decline toward the third week. Whereas, a relatively higher normoxic basal EPO protein level in aged mice did not show significant increase until seventh day of hypoxia, but showed significant upregulation in prolonged hypoxia. Hence, we confirmed that there is a progressively increased accumulation of EPO during chronic hypoxia in young and aged mouse kidney, and the EPO upregulation during hypoxia showed a similarity with the pattern of increase in hematocrit, which we have reported previously.

  2. 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

    冯睿; 范娟; 刘一辉; 钱召强; 魏春玲; 任维


    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.

  3. Stem cell antigen 2 expression in adult and developing mice.

    Antica, M; Wu, L; Scollay, R


    Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

  4. Sugar expressions on the vaginal epithelium in pregnant mice.

    Yasunaga, Youhei; Takeuchi, Takashi; Shimokawa, Tetsuya; Nabeta, Motowo; Matsuu, Aya; Asano, Atsushi; Ohta, Yasuhiko


    Sugar expressions were examined on the epithelium of both the middle portion of the vagina and the vaginal portion of the cervical canal (CC) in pregnant mice to understand the pathogenesis of bacterial infection in the female reproductive organ by using a panel of lectins. As a result, N-acetylglucosamine was positive before pregnant day (P) 7 but negative after P10 and at diestrus on both the vagina and the CC. In addition, some differences in sugar expressions were seen between them. These results suggest that sugar expressions on the mucosal surface would change not only site-specifically but also time-dependently, and these sugar differences indicate the possibility of the alteration of the settled bacterial species on the vaginal mucosa in pregnancy.

  5. Methanol teratogenicity in mutant mice with deficient catalase activity and transgenic mice expressing human catalase.

    Siu, Michelle T; Wiley, Michael J; Wells, Peter G


    The role of catalase in methanol (MeOH) teratogenesis is unclear. In rodents it both detoxifies reactive oxygen species (ROS) and metabolizes MeOH and its formic acid (FA) metabolite. We treated pregnant mice expressing either high (hCat) or low catalase activity (aCat), or their wild-type (WT) controls, with either MeOH (4g/kg ip) or saline. hCat mice and WTs were similarly susceptible to MeOH-initiated ophthalmic abnormalities and cleft palates. aCat and WT mice appeared resistant, precluding assessment of the developmental impact of catalase deficiency. Catalase activity was respectively increased at least 1.5-fold, and decreased by at least 35%, in hCat and aCat embryos and maternal livers. MeOH and FA pharmacokinetic profiles were similar among hCat, aCat and WT strains. Although the hCat results imply no ROS involvement, embryo culture studies suggest this may be confounded by maternal factors and/or a requirement for higher catalase activity in the hCat mice.

  6. Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

    D. Feder


    Full Text Available Erythropoietin (EPO has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1, and tumor necrosis factor-α (TNF-α was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11. On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16 and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09. These results may help to clarify some of the direct actions of EPO on skeletal muscle.

  7. Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

    Feder, D.; Rugollini, M.; Santomauro, A. Jr; Oliveira, L.P.; Lioi, V.P. [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Santos, R. dos; Ferreira, L.G.; Nunes, M.T.; Carvalho, M.H. [Universidade de São Paulo, Instituto de Ciências Biomédicas, São Paulo, SP (Brazil); Delgado, P.O.; Carvalho, A.A.S. [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Fonseca, F.L.A. [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Universidade Federal de São Paulo, Ambientais e Farmacêuticas, Instituto de Ciências Químicas, Diadema, SP (Brazil)


    Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.

  8. CNPase Expression in Olfactory Ensheathing Cells

    Christine Radtke


    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.

  9. Adrenal glucocorticoids regulate adipsin gene expression in genetically obese mice.

    Spiegelman, B M; Lowell, B; Napolitano, A; Dubuc, P; Barton, D; Francke, U; Groves, D L; Cook, K S; Flier, J S


    Adipsin expression at the protein and mRNA levels is greatly reduced in several distinct syndromes of obesity in the mouse: genetic obesity due to the db/db and ob/ob genes, and a chemically induced model secondary to neonatal exposure to monosodium glutamate. We considered first the possibility that the adipsin gene might be identical to the db or ob locus and the lowered expression of this protein might result from a mutation in this gene. We show here that the adipsin structural gene is located on chromosome 10 and hence is physically distinct from any obesity genes so far identified in the mouse. A major role for the adrenal gland and adrenal glucocorticoids in the aberrant regulation of adipsin in these models of obesity is indicated by several experiments. Adrenalectomy of the ob/ob mouse raises the circulating levels of adipsin protein and the amount of this mRNA in epididymal fat pads (5-fold), although neither is increased to the levels seen in lean controls. Exogenous administration of corticosterone completely blocks the effects of adrenalectomy on adipsin, suggesting that the effect of this endocrine ablation is through reduction of adrenal glucocorticoids. Corticosterone administration also causes suppression in the levels of adipsin mRNA and protein in lean mice, although this decrease is never as severe as that seen in obese mice. The effect of exogenous corticosterone in lean mice occurs within 2 days and hence is not secondary to the obesity which these hormones eventually elicit. These results indicate that glucocorticoids can regulate adipsin expression in vivo and strongly suggest that the hyperglucocorticoid state seen in certain obese models plays a significant role in lowering adipsin mRNA and protein levels. Quantitative analysis of these experiments suggests that other as yet unknown neuroendocrine factors also function to suppress adipsin in obesity.

  10. BPI700-Fcγ1700 chimeric gene expression and its protective effect in a mice model of the lethal E. coli infection


    Background Infections caused by gram-negative bacteria (GNB) often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 (AAV2)-bacteriacidal permeability increasing protein 700 (BPI700) -fragment crystallizable gamma one 700 (Fcγ1700) chimeric gene transferred mice against the minimal lethal dose (MLD) of E.coli and application of gene therapy for bacterial infection.Methods After AAV2-BPI700-Fcγ1700 virus transfection,dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells (CHO-K1cells). Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI700-Fcγ1700 gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit (CFU) count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine.Results BPI1-199-Fc(1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI700-Fc(1700 gene transferred mice (36.7%) was significantly higher than that of AAV2-enhanced green fluorescent protein (AAV2- EGFP) gene transferred mice (3.3%) and PBS control mice (5.6%). The survival rate of AAV2-BPI700-Fc(1700 gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine (tumor necrcsis factor-α and interleukin-1β) in serum of the AAV2-BPI

  11. Enhancement of naked FIX minigene expression by chloroquine in mice

    Hong-yan CHEN; Huan-zhang ZHU; Bin LU; Xuan XU; Ji-hua YAO; Qi SHEN; Jing-lun XUE


    AIM: To study the effect of chloroquine on the expression of human clotting factor IX (hFIX) in mice. METHODS:Hydrodynamics-based naked DNA plasmid administration was performed by tail vein injection of 10 μg of pCMVhFIX and chloroquine (0, 100, 200, and 500 μmol/L) in 2.2 mL of Ringer' solution within 6-7 s, the level and stability of hFIX expression, liver damage and toxicity were then examined. RESULTS: The maximum expression of hFIX level was 4.4±-1.8 mg/L at 8 h after injection, 9.7±1.6 mg/L at 24 h only existed in 200 μmol/L chloroquinetreated animals, which is 3-4 fold higher than that of control (P<0.01). There is no significant difference observed among all the treated groups, 3 d later. Transaminase level and liver histological study showed the damage of liver was not related to chloroquine (P>0.05). CONCLUSION: Chloroquine can enhance and sustain exogenous gene expression in vivo without side effect under our experimental conditions.

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

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie


    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.

  13. Intra-bone marrow-bone marrow transplantation slows disease progression and prolongs survival in G93A mutant SOD1 transgenic mice, an animal model mouse for amyotrophic lateral sclerosis.

    Ohnishi, Shizuo; Ito, Hidefumi; Suzuki, Yasuhiro; Adachi, Yasushi; Wate, Reika; Zhang, Jianhua; Nakano, Satoshi; Kusaka, Hirofumi; Ikehara, Susumu


    It has been reported that bone marrow transplantation (BMT) has clinical effects on not only hematopoietic diseases and autoimmune diseases but also solid malignant tumors and metabolic diseases. We have found that intra-bone marrow-bone marrow transplantation (IBM-BMT) is superior to conventional intravenous BMT, since IBM-BMT enables rapid recovery of donor hematopoiesis and reduces the extent of graft-versus-host disease (GVHD). In this experiment, we examined the effects of IBM-BMT on symptomatic G93A mutant SOD1 transgenic mice (mSOD1 Tg mice), a model mouse line for amyotrophic lateral sclerosis (ALS). Symptomatic mSOD1 Tg mice (12 weeks old) were irradiated with 6Gyx2 at a 4-hour interval, one day before IBM-BMT. The mice were transplanted with bone marrow cells (BMCs) from 12-wk-old eGFP-transgenic C57BL/6 mice (eGFP Tg mice) or BMCs from 12-wk-old mSOD1 Tg mice. The ALS model mice transplanted with BMCs from eGFP Tg mice showed longer survival and slower disease progression than those transplanted with BMCs from mSOD1 Tg mice or untreated mSOD1 Tg mice. There was a significantly high number of eGFP(+) cells in the anterior horn of the spinal cord of the mSOD1 Tg mice transplanted with BMCs of eGFP Tg mice, some of which expressed Iba-1, a marker of microglia, although they did not differentiate into neural cells. These results suggest that the replacement with normal hematopoietic cells improved the neural cell environment, thereby slowing the progression of the disease.

  14. Bacterial xylanase expression in mammalian cells and transgenic mice.

    Fontes, C M; Ali, S; Gilbert, H J; Hazlewood, G P; Hirst, B H; Hall, J


    The energy which simple-stomached livestock can derive from dietary plant material is limited by the lack of plant polysaccharide degrading enzymes in their gastro-intestinal (GI) tract and the inefficient microbial fermentation of such material in their hind-gut. In poultry the non-starch polysaccharides found in cereal grains can also impair normal digestive function as they form viscous gels in the GI tract inhibiting the breakdown and absorption of nutrients. The nutrition of such livestock could, therefore, be improved by the introduction of enzymes able to degrade plant polysaccharides in the small intestine. We describe the expression of a xylanase, XYLY', from the bacterium Clostridium thermocellum in mammalian cells and the exocrine pancreas of transgenic mice. The enzyme is synthesised, secreted and functionally active in the eukaryote system. This work demonstrates the feasibility of generating animals with the endogenous capacity to depolymerise the xylan component of hemi-cellulose.

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

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


    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.

  16. Decreased expression of natriuretic peptides associated with lipid accumulation in cardiac ventricle of obese mice

    Bartels, E.D.; Nielsen, J.M.; Bisgaard, L.S.


    /db, and fat-fed C57BL/6 mice as compared with their respective controls. The db/db and ob/ob mice displayed impaired cardiac function, whereas the fat-fed mice had almost normal cardiac function. Moreover, the ventricular expression of hypertrophic genes (a- and ß-myosin heavy chain and a...

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

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


    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.

  18. Delayed wound healing due to increased interleukin-10 expression in mice with lymphatic dysfunction.

    Kimura, Takayuki; Sugaya, Makoto; Blauvelt, Andrew; Okochi, Hitoshi; Sato, Shinichi


    Skin wound healing is an interactive process involving soluble mediators, ECM, resident cells, and infiltrating cells. Little is known about wound healing in the presence of lymphedema. In this study, we investigated wound healing using kCYC⁺/⁻ mice, which demonstrate severe lymphatic dysfunction. Wound healing was delayed significantly in kCYC⁺/⁻ mice when compared with WT mice. In wounded skin of kCYC⁺/⁻ mice, mast cell numbers were increased compared with WT mice, whereas macrophage numbers were decreased. Moreover, IL-10 expression by mast cells was increased, and expression of bFGF, mainly produced by macrophages, was decreased in wounded skin of kCYC⁺/⁻ mice compared with WT mice. We next crossed kCYC⁺/⁻ mice with IL-10⁻/⁻ mice, which were reported to show accelerated wound closure. In kCYC⁺/⁻ IL-10⁺/⁻ mice, time course of wound healing, numbers of macrophages, and IL-10 mRNA expression levels in wounded skin were comparable with WT IL-10⁺/⁻ mice. Similar results were obtained using a different lymphedema model, in which circumferential skin excision was performed on the tails of mice to remove the superficial lymphatics. In summary, these findings suggest that IL-10 plays an important role in delayed wound healing in the setting of lymphatic dysfunction.

  19. Calretinin-Periglomerular Interneurons in Mice Olfactory Bulb: Cells of Few Words

    Alex Fogli Iseppe; Angela Pignatelli; Ottorino Belluzzi


    Within the olfactory bulb (OB), periglomerular (PG) cells consist of various types of interneurons, generally classified by their chemical properties such as neurotransmitter and calcium binding proteins.Calretinin (CR) characterizes morphologically and functionally the more numerous and one of the less known subpopulation of PG cells in the OB. Using of transgenic mice expressing eGFP under the CR promoter, we have tried to obtain the first functional characterization of these cells. Electro...

  20. Effect of Oxytropis glabra DC. Poisoning on α-Mannosidase(AMA) Expression in Mice Brain Tissue

    Wang Shuai; Jia Qizhen; Zhang Ling; Chen Genyuan; Ma Chunhui


    The effect of Oxytropis glabra DC. on α-mannosidase( AMA) expression in mice brain tissue was explored to reveal the toxicity mechanism of O. glabra. Forty mice were randomly divided into four groups,namely control group,experimental group I,experimental group II and experimental group III. The mice in three experimental groups were fed with O. glabra at the doses of 1,5 and 10 g per kilogram weight,respectively. After challenge for 63 d,mice brains were collected to detect changes in distribution and expression of AMA in different brain regions. The results showed that O. glabra poisoning led to declined AMA mRNA expression in mice brain tissue,but the mice in experimental group I had no significant difference with those in control group( P > 0. 05). The AMA mRNA expression in cerebellum,cerebrum and thalamus of mice in experimental groups II and III were significantly lower than that in control group( P 0. 05). AMA had very weak expression in hippocampus and brainstem,but it had expressions in other regions,and the expression was positively correlated with the number of neurons and granulosa cells. The results showed that different doses of O. glabra reduced AMA mRNA expression in mice brain tissue,while cerebellum,cerebrum and thalamus were the main target function areas.

  1. Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice.

    Miriam Matamales

    Full Text Available Precise identification of neuronal populations is a major challenge in neuroscience. In the striatum, more than 95% of neurons are GABAergic medium-sized spiny neurons (MSNs, which form two intermingled populations distinguished by their projections and protein content. Those expressing dopamine D(1-receptors (D1Rs project preferentially to the substantia nigra pars reticulata (SNr, whereas those expressing dopamine D(2- receptors (D2Rs project preferentially to the lateral part of the globus pallidus (LGP. The degree of segregation of these populations has been a continuous subject of debate, and the recent introduction of bacterial artificial chromosome (BAC transgenic mice expressing fluorescent proteins driven by specific promoters was a major progress to facilitate striatal neuron identification. However, the fraction of MSNs labeled in these mice has been recently called into question, casting doubt on the generality of results obtained with such approaches. Here, we performed an in-depth quantitative analysis of striatal neurons in drd1a-EGFP and drd2-EGFP mice. We first quantified neuronal and non-neuronal populations in the striatum, based on nuclear staining with TO-PRO-3, and immunolabeling for NeuN, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein Mr approximately 32,000, and various markers for interneurons. TO-PRO-3 staining was sufficient to identify MSNs by their typical nuclear morphology and, with a good probability, interneuron populations. In drd1a-EGFP/drd2-EGFP double transgenic mice all MSNs expressed EGFP, which was driven in about half of them by drd1a promoter. Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%. In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP. Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons. It also

  2. Diurnal variation of hepatic antioxidant gene expression in mice.

    Yi-Qiao Xu

    Full Text Available BACKGROUND: This study was aimed to examine circadian variations of hepatic antioxidant components, including the Nrf2- pathway, the glutathione (GSH system, antioxidant enzymes and metallothionein in mouse liver. METHODS AND RESULTS: Adult mice were housed in light- and temperature-controlled facilities for 2 weeks, and livers were collected every 4 h during the 24 h period. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. Hepatic mRNA levels of Nrf2, Keap1, Nqo1 and Gclc were higher in the light-phase than the dark-phase, and were female-predominant. Hepatic GSH presented marked circadian fluctuations, along with glutathione S-transferases (GST-α1, GST-µ, GST-π and glutathione peroxidase (GPx1. The expressions of GPx1, GST-µ and GST-π mRNA were also higher in females. Antioxidant enzymes Cu/Zn superoxide dismutase (Sod1, catalase (CAT, cyclooxygenase-2 (Cox-2 and heme oxygenase-1 (Ho-1 showed circadian rhythms, with higher expressions of Cox-2 and CAT in females. Metallothionein, a small non-enzymatic antioxidant protein, showed dramatic circadian variation in males, but higher expression in females. The circadian variations of the clock gene Brain and Muscle Arnt-like Protein-1(Bmal1, albumin site D-binding protein (Dbp, nuclear receptor Rev-Erbα (Nr1d1, period protein (Per1 and Per2 and cryptochrome 1(Cry1 were in agreement with the literature. Furthermore, acetaminophen hepatotoxicity is more severe when administered in the afternoon when hepatic GSH was lowest. CONCLUSIONS: Circadian variations and gender differences in transcript levels of antioxidant genes exist in mouse liver, which could affect body responses to oxidative stress at different times of the day.

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

    Quatromoni Jon G


    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.

  4. Effects of Aging and Advanced Glycation on Gene Expression in Cerebrum and Spleen of Mice



    Objective To analyze the effects of aging or advanced glycation on gene expression in the cerebrum and spleen of female C57BL/6J mice. Methods The gene expression profile was determined by using cDNA expression arrays containing 588 cDNA. Results Aging and advanced glycation resulted in differential gene expression patterns of cerebrum and spleen compared with young mice. Among the 80 genes detected in cerebrum, 43 exhibited a change in mRNA ratios with aging or treatment. Thirty-four changes (79%) were common in aged and D-galactose treated mice,whereas the cerebrum from aged and AGE-lysine treated mice showed common changes in expression of 38 genes(88%). Of the 86 genes detected in spleen, 29 (34%) displayed an age-related decrease in expression, whereas 3 (3%) displayed an increase in expression levels with aging. Eighteen genes from the detectable genes exhibited expression changes in both cerebrum and spleen of mice.Conclusions The gene expression profiles of D-galactose and AGE-lysine treated mice resemble those of aged mice. Use of cDNA hybridization arrays may provide a promising tool to explore the mechanism of aging at a molecular level.

  5. 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

    乔宇; 白荣平; 盛玉彪


    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。

  6. Gene expression of LOX-1, VCAM-1, and ICAM-1 in pre-atherosclerotic mice

    Fisker Hag, Anne Mette; Pedersen, Sune Folke; Kjaer, Andreas


    -atherosclerotic mice. Furthermore, the plasma levels of the soluble VCAM-1 and ICAM-1 were compared to the gene expression profiles. Gene expressions of LOX-1 and VCAM-1 were up-regulated in young apoE(-/-) mice, and thus, it seems probable that these genes play a role in pre-atherosclerosis. Contrarily, the gene...... expression profile of ICAM-1 did not show any apparent differences between the groups, questioning the involvement of this molecule in the early development of atherosclerosis. Plasma levels of sVCAM-1 and sICAM-1 were similar in all mice and did not correlate with the vascular gene expression...

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

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


    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.)

  8. 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

    周前进; 姜小磊; 张红丽; 杜爱芳


    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.

  9. 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

    李拓凡; 多婷; 梁雄燕; 顾玉芳; 杨玉莹


    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.

  10. REM sleep deprivation increases the expression of interleukin genes in mice hypothalamus.

    Kang, Won Sub; Park, Hae Jeong; Chung, Joo-Ho; Kim, Jong Woo


    Recently, evidence has suggested the possible involvement of inflammatory cytokines in sleep deprivation (SD). In this study, we assessed the patterns of inflammatory gene regulation in the hypothalamus of REM SD mice. C57BL/6 mice were randomly assigned to two groups, SD (n=15) and control groups (n=15). Mice in the SD group were sleep-deprived for 72h using modified multiple platforms. Microarray analysis on inflammatory genes was performed in mice hypothalamus. In addition, interleukin 1 beta (IL1β) protein expression was analyzed by the immunochemistry method. Through microarray analysis, we found that expressions of IL subfamily genes, such as IL1β (2.55-fold), IL18 (1.92-fold), IL11 receptor alpha chain 1 (1.48-fold), IL5 (1.41-fold), and IL17E genes (1.31-fold), were up-regulated in the hypothalamus of SD mice compared to the control. The increase in the expression of these genes was also confirmed by RT-PCR. Among these genes, the expression of IL1β was particularly increased in the hypothalamus of SD mice. Interestingly, we found that the protein expression of endogenous IL1β was also elevated in the hypothalamus of SD mice compared to the control mice. These results implicate that IL subfamily genes, and in particular, IL1β, may play a role in sleep regulation in the hypothalamus of REM SD mice.




    Introduction We investigated the extent to which intercellular adhesion molecule-1 (ICAM-1), a critical protein of the inflammatory response, is expressed in skeletal muscles of mdx mice (a murine model of Duchenne muscular dystrophy). Methods Muscles were collected from control and mdx mice at 2–24 weeks of age and analyzed for ICAM-1 expression by means of Western blot and immunofluorescence. Results Western blot revealed higher expression of ICAM-1 in mdx compared with control muscles through 24 weeks of age. In contrast to control muscles, ICAM-1 was expressed on the membrane of damaged, regenerating, and normal myofibers of mdx mice. CD11b+ myeloid cells also expressed ICAM-1 in mdx muscles, and CD11b+ cells were closely associated with the membrane of myofibers expressing ICAM-1. Conclusions These findings support a paradigm in which ICAM-1 and its localization to myofibers in muscles of mdx mice contributes to the dystrophic pathology. PMID:25728314

  12. Gnotobiotic IL-10; NF-kappaB mice develop rapid and severe colitis following Campylobacter jejuni infection.

    Elisabeth Lippert

    Full Text Available Limited information is available on the molecular mechanisms associated with Campylobacter jejuni (C. jejuni induced food-borne diarrheal illnesses. In this study, we investigated the function of TLR/NF-kappaB signaling in C. jejuni induced pathogenesis using gnotobiotic IL-10(-/-; NF-kappaB(EGFP mice. In vitro analysis showed that C. jejuni induced IkappaB phosphorylation, followed by enhanced NF-kappaB transcriptional activity and increased IL-6, MIP-2alpha and NOD2 mRNA accumulation in infected-mouse colonic epithelial cells CMT93. Importantly, these events were blocked by molecular delivery of an IkappaB inhibitor (Ad5IkappaBAA. NF-kappaB signalling was also important for C.jejuni-induced cytokine gene expression in bone marrow-derived dendritic cells. Importantly, C. jejuni associated IL-10(-/-; NF-kappaB(EGFP mice developed mild (day 5 and severe (day 14 ulcerating colonic inflammation and bloody diarrhea as assessed by colonoscopy and histological analysis. Macroscopic analysis showed elevated EGFP expression indicating NF-kappaB activation throughout the colon of C. jejuni associated IL-10(-/-; NF-kappaB(EGFP mice, while fluorescence microscopy revealed EGFP positive cells to be exclusively located in lamina propria mononuclear cells. Pharmacological NF-kappaB inhibition using Bay 11-7085 did not ameliorate C. jejuni induced colonic inflammation. Our findings indicate that C. jejuni induces rapid and severe intestinal inflammation in a susceptible host that correlates with enhanced NF-kappaB activity from lamina propria immune cells.

  13. 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

    陈艳春; 王俊; 王士礼; 蔡昌枰; 李彪; 张一帆; 郭睿


    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

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

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


    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.

  15. Radioprotective effects of the expression of FLT3 ligand regulated by Egr-1 regulated element on radiation injury of SCID mice

    Du Nan; Pei Xuetao; Luo Chengji; Su Yongping; Cheng Tianmin


    Objective:In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transduced cell clones (HFCL/EF) had been selected by the addition of G418. The cells were exposed to γ-radiation by 60 Co source for 0.5-20Gy. The expressions of transduced cells were detected with FACS, Northern blot ELISA and CFU assay. The HFCL/EF and CD34+ cells from human umbilical cord blood were one after the other transplanted i.v. into sublethally irradiated severe combined immunodeficient (SCID) mice. The white blood cell amount in peripheral blood and human cell engrafted in recipent mice were detected by flow cytometry and CFU-GM etc. Results:The activity of EGFP in transduced cells increased by 3.1 fold as compared to non-transduced cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605.46±107.21pg/ml, which were significantly higher than the control group (214.45±35.61pg/ml). The effects of FL in HFCL/EF cultural supernatants on expansion of CD34+ cells derived from cord blood in the presence of SCF, IL-6 and IL-3 were also studied. The results showed that at day 10 of culture the number of CD34+ cells increased by 173. 09±11.58×103/ml, which was significantly higher than that of non-radiation group(68. 04± 13. 73 × 103/ml). It showed that radiation can enhance the ability of the supernatants containing FL of HFCL/EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects. The HFCL/EF and CD34

  16. Cardiomyocyte-specific expression of lamin a improves cardiac function in Lmna-/- mice.

    Richard L Frock

    Full Text Available Lmna(-/- mice display multiple tissue defects and die by 6-8 weeks of age reportedly from dilated cardiomyopathy with associated conduction defects. We sought to determine whether restoration of lamin A in cardiomyocytes improves cardiac function and extends the survival of Lmna(-/- mice. We observed increased total desmin protein levels and disorganization of the cytoplasmic desmin network in ~20% of Lmna(-/- ventricular myocytes, rescued in a cell-autonomous manner in Lmna(-/- mice expressing a cardiac-specific lamin A transgene (Lmna(-/-; Tg. Lmna(-/-; Tg mice displayed significantly increased contractility and preservation of myocardial performance compared to Lmna(-/- mice. Lmna(-/-; Tg mice attenuated ERK1/2 phosphorylation relative to Lmna(-/- mice, potentially underlying the improved localization of connexin43 to the intercalated disc. Electrocardiographic recordings from Lmna(-/- mice revealed arrhythmic events and increased frequency of PR interval prolongation, which is partially rescued in Lmna(-/-; Tg mice. These findings support our observation that Lmna(-/-; Tg mice have a 12% median extension in lifespan compared to Lmna(-/- mice. While significant, Lmna(-/-; Tg mice only have modest improvement in cardiac function and survival likely stemming from the observation that only 40% of Lmna(-/-; Tg cardiomyocytes have detectable lamin A expression. Cardiomyocyte-specific restoration of lamin A in Lmna(-/- mice improves heart-specific pathology and extends lifespan, demonstrating that the cardiac pathology of Lmna(-/- mice limits survival. The expression of lamin A is sufficient to rescue certain cellular defects associated with loss of A-type lamins in cardiomyocytes in a cell-autonomous fashion.

  17. Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

    Park, Young Joo [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Seoul National University Bundang Hospital, Seoul (Korea, Republic of); Lee, Eun Kyung [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Lee, Yoon Kwang [Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Park, Do Joon; Jang, Hak Chul [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Moore, David D., E-mail: [Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States)


    Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR{sup −/−} mice, but not in hypothyroid PXR{sup −/−} mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR{sup −/−} hypothyroid mice, and this induction was abolished in CAR{sup −/−} mice and in and CAR{sup −/−} PXR{sup −/−} double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR{sup −/−} mice and lowest in WT and PXR{sup −/−} mice. Hypothyroid WT or PXR{sup −/−} mice survived chronic CBZ treatment, but all hypothyroid CAR{sup −/−} and CAR{sup −/−} PXR{sup −/−} mice died, with CAR{sup −/−}PXR{sup −/−} mice surviving longer than CAR{sup −/−} mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge. -- Highlights: ► Hypothyroid status activates CAR in mice and induces Cyp2b10 expression. ► Hypothyroid status suppresses PXR activity in mice and represses Cyp3a11 expression. ► These responses balance each other out in normal mice.

  18. Monitoring immediate-early gene expression through firefly luciferase imaging of HRS/J hairless mice

    Geusz Michael E


    Full Text Available Abstract Background Gene promoters fused to the firefly luciferase gene (luc are useful for examining gene regulation in live transgenic mice and they provide unique views of functioning organs. The dynamics of gene expression in cells and tissues expressing luciferase can be observed by imaging this enzyme's bioluminescent oxidation of luciferin. Neural pathways involved in specific behaviors have been identified by localizing expression of immediate-early genes such as c-fos. A transgenic mouse line with luc controlled by the human c-fos promoter (fos::luc has enabled gene expression imaging in brain slice cultures. To optimize imaging of immediate-early gene expression throughout intact mice, the present study examined fos::luc mice and a second transgenic mouse containing luc controlled by the human cytomegalovirus immediate-early gene 1 promoter and enhancer (CMV::luc. Because skin pigments and hair can significantly scatter light from underlying structures, the two transgenic lines were crossed with a hairless albino mouse (HRS/J to explore which deep structures could be imaged. Furthermore, live anesthetized mice were compared with overdosed mice. Results Bioluminescence imaging of anesthetized mice over several weeks corresponded with expression patterns in mice imaged rapidly after a lethal overdose. Both fos::luc and CMV::luc mice showed quantifiable bright bioluminescence in ear, nose, paws, and tail whether they were anesthetized or overdosed. CMV::luc and fos::luc neonates had bioluminescence patterns similar to those of adults, although intensity was significantly higher in neonates. CMV::luc mice crossed with HRS/J mice had high expression in bone, claws, head, pancreas, and skeletal muscle, but less in extremities than haired CMV::luc mice. Imaging of brain bioluminescence through the neonatal skull was also practical. By imaging luciferin autofluorescence it was clear that substrate distribution did not restrict bioluminescence

  19. Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm].

    Kokoza, V; Ahmed, A; Wimmer, E A; Raikhel, A S


    We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G2-G6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5' upstream region.

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

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


    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...

  1. PGC-1alpha mediates exercise-induced skeletal muscle VEGF expression in mice

    Leick, Lotte; Hellsten, Ylva; Fentz, Joachim


    The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO) and litterm......The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO......) and littermate wild-type (WT) mice were submitted to either 1) 5 wk of exercise training, 2) lifelong (from 2 to 13 mo of age) exercise training in activity wheel, 3) a single exercise bout, or 4) 4 wk of daily subcutaneous AICAR or saline injections. In skeletal muscle of PGC-1alpha KO mice, VEGF protein...... expression was approximately 60-80% lower and the capillary-to-fiber ratio approximately 20% lower than in WT. Basal VEGF mRNA expression was similar in WT and PGC-1alpha KO mice, but acute exercise and AICAR treatment increased the VEGF mRNA content in WT mice only. Exercise training of young mice increased...

  2. Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice


    The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encoding human erythropoietin (hEPO) and governed by regulatory sequences of mouse whey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%. hEPO was expressed in the milk of 16 mice of 39 mice measured by hEPO ELISA kit .The expression level gets over 15 m g/mL.

  3. 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

    刘彦; 曾庆仁; 杨胜辉; 魏琦; 周军; 李立新; 兰玲梅


    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.

  4. The progesterone and estrogen modify the uterine prolactin and prolactin receptor expression of hyperprolactinemic mice.

    do Amaral, Vinícius Cestari; Carvalho, Kátia Candido; Maciel, Gustavo Arantes Rosa; Simoncini, Tommaso; da Silva, Priscilla Ludovico; Marcondes, Rodrigo Rodrigues; Soares, José Maria; Baracat, Edmund Chada


    The aim of this study was to evaluate the effects of metoclopramide-induced hyperprolactinemia on the prolactin (PRL) and PRL receptor's expression in the uterus of mice. For this purpose, 49 Swiss mice were divided into the following groups: GrSS (non-ovariectomized mice given vehicle); GrMET (non-ovariectomized mice treated with metoclopramide); OvSS (ovariectomized mice given vehicle); OvMET (ovariectomized mice treated with metoclopramide); OvMET+17βE (ovariectomized mice treated with metoclopramide and 17β estradiol); OvMET+MP (ovariectomized mice treated with metoclopramide and micronized progesterone); OvMET+17βE+MP (ovariectomized mice treated with metoclopramide and a solution of 17β estradiol and micronized progesterone). Immunohistochemical analyzes were evaluated semi-quantitatively. Our results showed that GrMET, OvMET+MP, and OvMET+17βE+MP presented strong PRL expression. OvMET and OvMET+17βE presented mild reaction, while GrSS and OvSS presented weak reaction. Concerning PRL receptor, OvMET+MP and OvMET+17βE+MP showed strong reaction; GrMET, OvSS, and OvMET+17βE showed mild reaction; and GrSS and OvMET showed weak reaction. These findings suggest that progesterone alone or in combination with estrogen may increase the expression of uterine PRL and PRL receptor.

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

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


    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.

  6. Optimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice.

    Abdul Mannan Baru

    Full Text Available Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells mice have greatly helped in exploring the biology of Foxp3(+ Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3(+ Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3(+ iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4(+Foxp3(+ iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP(+Foxp3(- cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP(- cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP(+Foxp3(+ iTregs exhibited potent suppressive activity comparable to that of natural eGFP(+Foxp3(+ Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP(+Foxp3(+ iTregs by use of DEREG mice. Isolation of functional Foxp3(+ iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP

  7. Expression of angiogenic regulators and skeletal muscle capillarity in selectively bred high aerobic capacity mice.

    Audet, Gerald N; Meek, Thomas H; Garland, Theodore; Olfert, I Mark


    Selective breeding for high voluntary wheel running in untrained mice has resulted in a 'mini muscle' (MM) phenotype, which has increased skeletal muscle capillarity compared with muscles from non-selected control lines. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) are essential mediators of skeletal muscle angiogenesis; thus, we hypothesized that untrained MM mice with elevated muscle capillarity would have higher basal VEGF expression and lower basal TSP-1 expression, and potentially an exaggerated VEGF response to acute exercise. We examined skeletal muscle morphology and skeletal muscle protein expression of VEGF and TSP-1 in male mice from two (untrained) mouse lines selectively bred for high exercise capacity (MM and Non-MM), as well as one non-selected control mouse line (normal aerobic capacity). In the MM mice, gastrocnemius (GA) and plantaris (PLT) muscle capillarity (i.e. capillary-to-fibre ratio and capillary density) were greater compared with control mice (P capillarity in PLT was greater than in control mice (P capillarity among groups. In the GA, MM mice had 58% greater basal VEGF (P capillarity is associated with altered balance between positive and negative angiogenic regulators (i.e. VEGF versus TSP-1, respectively). Based on the greater capillarity and significant VEGF response to exercise in MM mice, these data suggest that VEGF expression may, at least in part, be genetically determined.

  8. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica*


    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Ampli...

  9. Expression and Bioinformatics Analysis of SPACA4 in Human and Mice

    Ai-fa TANG; Zhen-dong YU; Yao-ting GUI; Xin GUO; Xian-xin LI; Wei-xiang LIU; Hui ZHU; Zhi-ming CAI


    Objective To analyze the expression of SPACA4 in human and mice. Methods Testes cRNA samples from Balb/c mice of different postnatal days were performed with mouse affymetrix chip to screen the expression of SPACA4 in mice. Sub-quantitative RT-PCR and bioinformatic tools were used here to describe the expression profile of SPACA4 in mice and human. Results The results of gene chip analysis indicated that the expression of mSPACA4 began after d 35 of postnatal testis in mice. Sub-quantitative RT-PCR assay showed that SPACA4 gene expressed exclusively in mouse and human testis, and mouse mSPACA4 gene expressed after d 35 of postnatal testis that was consistency with the results of gene chip analysis. By bioinformatics analysis, mSPACA4 is located in cell membrane (34.8%) or plasma membrane (34.8%), the signal peptide cleavage site between position 19 and 20 amino acids, transmembrane region between 2-20 and 101-126 amino acids, respectively, on mSPACA4 protein. Conclusion mSPACA4 and hSPACA4 were testis-specific genes, and the expression of mSPACA4 begins after d 35 of postnatal testis in mice. SPACA4 is a candidate for targeting in a sperm-based contraceptive vaccine.

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

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


    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.

  11. Hepatic temporal gene expression profiling in Helicobacter hepaticus-infected A/JCr mice.

    Boutin, Samuel R; Rogers, Arlin B; Shen, Zeli; Fry, Rebecca C; Love, Jennifer A; Nambiar, Prashant R; Suerbaum, Sebastian; Fox, James G


    Helicobacter hepaticus infection of A/JCr mice is a model of infectious liver cancer. We monitored hepatic global gene expression profiles in H. hepaticus infected and control male A/JCr mice at 3 months, 6 months, and 1 year of age using an Affymetrix-based oligonucleotide microarray platform on the premise that a specific genetic expression signature at isolated time points would be indicative of disease status. Model based expression index comparisons generated by dChip yielded consistent profiles of differential gene expression for H. hepaticus infected male mice with progressive liver disease versus uninfected control mice within each age group. Linear discriminant analysis and principal component analysis allowed segregation of mice based on combined age and lesion status, or age alone. Up-regulation of putative tumor markers correlated with advancing hepatocellular dysplasia. Transcriptionally down-regulated genes in mice with liver lesions included those related to peroxisome proliferator, fatty acid, and steroid metabolism pathways. In conclusion, transcriptional profiling of hepatic genes documented gene expression signatures in the livers of H. hepaticus infected male A/JCr mice with chronic progressive hepatitis and preneoplastic liver lesions, complemented the histopathological diagnosis, and suggested molecular targets for the monitoring and intervention of disease progression prior to the onset of hepatocellular neoplasia.

  12. Endothelial Expression of Scavenger Receptor Class B, Type I Protects against Development of Atherosclerosis in Mice

    Boris L. Vaisman


    Full Text Available The role of scavenger receptor class B, type I (SR-BI in endothelial cells (EC was examined in several novel transgenic mouse models expressing SR-BI in endothelium of mice with normal C57Bl6/N, apoE-KO, or Scarb1-KO backgrounds. Mice were also created expressing SR-BI exclusively in endothelium and liver. Endothelial expression of the Tie2-Scarb1 transgene had no significant effect on plasma lipoprotein levels in mice on a normal chow diet but on an atherogenic diet, significantly decreased plasma cholesterol levels, increased plasma HDL cholesterol (HDL-C levels, and protected mice against atherosclerosis. In 8-month-old apoE-KO mice fed a normal chow diet, the Tie2-Scarb1 transgene decreased aortic lesions by 24%. Mice expressing SR-BI only in EC and liver had a 1.5 ± 0.1-fold increase in plasma cholesterol compared to mice synthesizing SR-BI only in liver. This elevation was due mostly to increased HDL-C. In EC culture studies, SR-BI was found to be present in both basolateral and apical membranes but greater cellular uptake of cholesterol from HDL was found in the basolateral compartment. In summary, enhanced expression of SR-BI in EC resulted in a less atherogenic lipoprotein profile and decreased atherosclerosis, suggesting a possible role for endothelial SR-BI in the flux of cholesterol across EC.

  13. Altered nuclear factor-kappaB inducing kinase expression in insulin-resistant mice

    SU Lei; XIU Ling-ling; WEI Guo-hong; ZHONG Xing; LIU Yuan-yuan; CAO Xiao-pei; LI Yan-bing; XIAO Hai-peng


    Background Insulin resistance is an underlying feature of both type 2 diabetes and metabolic syndrome.Currently,it is unclear whether nuclear factor (NF)-κB inducing kinase (NIK) plays a role in the development of insulin resistance.The present in vivo study investigated the roles of NIK and IKB kinase α (IKKα) in obesity-induced insulin resistance using animal models.Methods NIK expression was evaluated by Westem blotting in male Lepob mice and C57BL/6J mice fed a high-fat diet (HFD) (45% fat).After metformin and sulfasalazine treatment,NIK expression was investigated during the improvement of insulin resistance.Results NIK was increased by about 1-fold in the renal tissues of Lepob mice and C57BL/6J mice fed a HFD for 12 weeks.After 1 and 3 weeks of high-fat feeding,we observed an almost 50% decrease in NIK and IKKα expression in the liver and renal tissues of C57BL/6J mice.NIK expression was significantly lower in the liver and renal tissues of HFD-fed mice that were treated with insulin sensitizers,metformin and sulfasalazine.However,IKKα expression was increased after metformin treatment in both tissues.Conclusion These results suggest a possible role of NIK in the liver and renal tissues of insulin-resistant mice.

  14. Dipeptidyl peptidase expression during experimental colitis in mice

    Yazbeck, Roger; Sulda, Melanie L; Howarth, Gordon S


    We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection.......We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection....

  15. Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

    Chen, Zhiqi; Yu, Kai; Zhu, Fang; Gorczynski, Reginald


    Background and aim CD200:CD200 receptor (CD200R) interactions lead to potent immunosuppression and inhibition of autoimmune inflammation. We investigated the effect of "knockout"of CD200 or CD200R, or over-expression of CD200, on susceptibility to dextran sodium sulfate (DSS)—induced colitis, a mouse model of inflammatory bowel disease (IBD). Methods Acute or chronic colitis was induced by administration of dextran sodium sulfate (DSS) in four groups of age-matched C57BL/6 female mice: (1) CD200-transgenic mice (CD200tg); (2) wild-type (WT) mice; (3) CD200 receptor 1-deficient (CD200R1KO) mice; and (4) CD200-deficient (CD200KO) mice. The extent of colitis was determined using a histological scoring system. Colon tissues were collected for quantitative RT-PCR and Immunohistochemical staining. Supernatants from colonic explant cultures and mononuclear cells isolated from colonic tissue were used for ELISA. Results CD200KO and CD200R1KO mice showed greater sensitivity to acute colitis than WT mice, with accelerated loss of body weight, significantly higher histological scores, more severe infiltration of macrophages, neutrophils and CD3+ cells, and greater expression of macrophage-derived inflammatory cytokines, whose production was inhibited in vitro (in WT/CD200KO mouse cells) by CD200. In contrast, CD200tg mice showed less sensitivity to DSS compared with WT mice, with attenuation of all of the features seen in other groups. In a chronic colitis model, greater infiltration of Foxp3+ regulatory T (Treg) cells was seen in the colon of CD200tg mice compared to WT mice, and anti-CD25 mAb given to these mice attenuated protection. Conclusions The CD200:CD200R axis plays an immunoregulatory role in control of DSS induced colitis in mice. PMID:26841120

  16. DNA-encapsulated magnesium phosphate nanoparticles elicit both humoral and cellular immune responses in mice

    Gajadhar Bhakta


    Full Text Available The efficacy of pEGFP (plasmid expressing enhanced green fluorescent protein-encapsulated PEGylated (meaning polyethylene glycol coated magnesium phosphate nanoparticles (referred to as MgPi-pEGFP nanoparticles for the induction of immune responses was investigated in a mouse model. MgPi-pEGFP nanoparticles induced enhanced serum antibody and antigen-specific T-lymphocyte responses, as well as increased IFN-γ and IL-12 levels compared to naked pEGFP when administered via intravenous, intraperitoneal or intramuscular routes. A significant macrophage response, both in size and activity, was also observed when mice were immunized with the nanoparticle formulation. The response was highly specific for the antigen, as the increase in interaction between macrophages and lymphocytes as well as lymphocyte proliferation took place only when they were re-stimulated with recombinant green fluorescence protein (rGFP. Thus the nanoparticle formulation elicited both humoral as well as cellular responses. Cytokine profiling revealed the induction of Th-1 type responses. The results suggest DNA-encapsulated magnesium phosphate (MgPi nanoparticles may constitute a safer, more stable and cost-efficient DNA vaccine formulation.

  17. Male and female NOD mice differentially express peroxisome proliferator-activated receptors and pathogenic cytokines.

    Yaacob, Nik Soriani; Goh, Kenny Soen Keong; Norazmi, Mohd Nor


    The peroxisome proliferator-activated receptors (PPARs) have been implicated in regulating the immune response. We determined the relative changes in the transcriptional expression of PPAR isoforms (α, γ1 and γ2) and cytokines involved in the pathogenesis of type 1 diabetes (T1D) in the immune cells of 5 weeks, 10 weeks and diabetic male non-obese diabetic (NOD) mice compared to those of female NOD mice from our previous studies, "normalized" against their respective non-obese diabetic resistant (NOR) mice controls. Overall PPARα was significantly more elevated in the macrophages of female NOD mice of all age groups whereas PPARγ, particularly the PPARγ2 isoform was more depressed in the macrophages and CD4(+) lymphocytes of female NOD mice compared to their male counterparts. The pro-inflammatory cytokines, IL-1 and TNFα, as well as the Th1 cytokines, IL-2 and IFNγ were more elevated in female NOD mice whereas the Th2 cytokine, IL-4, was more depressed in these mice compared to their male counterparts. These findings suggest that the preponderance of T1D in female NOD mice may be influenced by the more pronounced changes in the expression of PPAR isoforms and pathogenic cytokines compared to those in male NOD mice. Copyright © 2010 Elsevier GmbH. All rights reserved.

  18. In vivo modulation of epidermal growth factor receptor phosphorylation in mice expressing different gangliosides.

    Daniotti, Jose L; Crespo, Pilar M; Yamashita, Tadashi


    We studied in this work the in vivo phosphorylation of the epidermal growth factor receptor (EGFr) in skin from knockout mice lacking different ganglioside glycosyltransferases. Results show an enhancement of EGFr phosphorylation, after EGF stimulation, in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice as compared with wild-type and Sial-T1 knockout mice. Qualitative analysis of ganglioside composition in mice skin suggest that the increase of EGFr phosphorylation observed in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice in response to EGF might not be primary attributed to the expression of GD3 or a-series gangliosides in mice skin. These studies provide, for the first time, an approach for studying the molecular mechanisms involved in the in vivo regulation of EGFr function by gangliosides.

  19. Differential Expression of Long Noncoding RNAs between Sperm Samples from Diabetic and Non-Diabetic Mice

    Guang-Jian Jiang; Teng Zhang; Tian An; Dan-Dan Zhao; Xiu-Yan Yang; Dong-Wei Zhang; Yi Zhang; Qian-Qian Mu; Na Yu; Xue-Shan Ma; Si-Hua Gao


    To investigate the potential core reproduction-related genes associated with the development of diabetes, the expression profiles of long noncoding RNA (lncRNA) and messenger RNA (mRNA) in the sperm of diabetic mice were studied. We used microarray analysis to detect the expression of lncRNAs and coding transcripts in six diabetic and six normal sperm samples, and differentially expressed lncRNAs and mRNAs were identified through Volcano Plot filtering. The function of differentially expresse...

  20. 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

    胡锦辉; 邱潮林; 华玲; 谢秋华; 何小雪; 沈振华; 胡珺


    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

  1. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.

    Kamioka, Yuji; Sumiyama, Kenta; Mizuno, Rei; Sakai, Yoshiharu; Hirata, Eishu; Kiyokawa, Etsuko; Matsuda, Michiyuki


    Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

  2. The expression of nerve growth factor in mice lung following low-level toluene exposure.

    Fujimaki, Hidekazu; Tin-Tin-Win-Shwe; Yamamoto, Shoji; Nakajima, Daisuke; Goto, Sumio


    To clarify the effect of indoor air pollutants on nerve growth factor (NGF) production in lung, male C3H/HeN mice were exposed to filtered air (control) or toluene at levels of 0.9 ppm, 9 ppm, or 90 ppm for 30 min via nose-only inhalation on days 0, 1, 2, 7, 14, 21, 28, 35, 42, 49 and 56. As an allergic mouse model, some mice (n=24) were immunized with ovalbumin. Lungs from each mouse were collected to determine NGF and related receptor expressions using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. NGF and TrkA mRNAs were increased in the lungs of the immunized mice following exposure to 9 ppm toluene (n=6) (Ptoluene-exposed, immunized mice. To determine NGF mediating signaling, we also examined mRNA expression of neurotrophin receptor p75 (p75(NTR)) and oxidative stress marker, heme oxygenase (HO)-1 in the lung. There is no difference in the expressions of p75(NTR) and HO-1 between toluene-exposed and control mice. The expression of CCL2 and CCL3 mRNAs was significantly elevated in 9 ppm toluene-exposed, immunized mice. These findings suggest that the exposure with volatile organic compounds enhanced NGF expression and airway inflammation stronger in allergic individuals than in healthy individuals.

  3. Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice

    Callow, M.J.; Stoltzfus, L.J.; Rubin, E.M. [Lawrence Berkeley Lab., CA (United States); Lawn, R.M. [Stanford Univ., CA (United States)


    The atherogenic macromolecule lipoprotein(a) [Lp(a)] has resisted in vivo analyses partly because it is found in a limited number of experimental animals. Although transgenic mice expressing human apolipoprotein (a) [apo(a)] have previously been described, they failed to assemble Lp(a) particles because of the inability of human apo(a) to associate with mouse apolipoprotein B (apoB). The authors isolated a 90-kilobase P1 phagemid containing the human apoB gene and with this DNA generated 13 lines of transgenic mice of which 11 expressed human apoB. The human apoB transcript was expressed and edited in the liver of the transgenic mice. Plasma concentrations of human apoB, as well as low density lipoprotein (LDL), were related to transgene copy number; the transgenic line with the most copies of human apoB had a >4-fold increase in LDL cholesterol compared with nontransgenics and a lipoprotein profile similar to that of humans. When human apoB and apo(a) transgenic mice were bred together, plasma apo(a) in mice expressing both human proteins was tightly associated with lipoproteins in the LDL density region. These studies demonstrate the successful expression of human apoB and the efficient assembly of Lp(a) in mice.

  4. Effects of Repeated Ethanol Exposures on NMDA Receptor Expression and Locomotor Sensitization in Mice Expressing Ethanol Resistant NMDA Receptors

    den Hartog, Carolina R.; Gilstrap, Meghin; Eaton, Bethany; Lench, Daniel H.; Mulholland, Patrick J.; Homanics, Gregg. E.; Woodward, John J.


    Evidence from a large number of preclinical studies suggests that chronic exposure to drugs of abuse, such as psychostimulants or ethanol induces changes in glutamatergic transmission in key brain areas associated with reward and control of behavior. These changes include alterations in the expression of ionotropic glutamate receptors including N-methyl-D-aspartate receptors (NMDAR) that are important for regulating neuronal activity and synaptic plasticity. NMDA receptors are inhibited by ethanol and reductions in NMDA-mediated signaling are thought to trigger homestatic responses that limit ethanol's effects on glutamatergic transmission. Following repeated exposures to ethanol, these homeostatic responses may become unstable leading to an altered glutamatergic state that contributes to the escalations in drinking and cognitive deficits observed in alcohol-dependent subjects. An important unanswered question is whether ethanol-induced changes in NMDAR expression are modulated by the intrinsic sensitivity of the receptor to ethanol. In this study, we examined the effects of ethanol on NMDAR subunit expression in cortical (orbitofrontal, medial prefrontal), striatal (dorsal and ventral striatum) and limbic (dorsal hippocampus, basolateral amygdala) areas in mice genetically modified to express ethanol-resistant receptors (F639A mice). These mice have been previously shown to drink more ethanol than their wild-type counterparts and have altered behavioral responses to certain actions of ethanol. Following long-term voluntary drinking, F639A mice showed elevations in GluN2A but not GluN1 or GluN2B expression as compared to wild-type mice. Mice treated with repeated injections with ethanol (2–3.5 g/kg; i.p.) showed changes in NMDAR expression that varied in a complex manner with genotype, brain region, subunit type and exposure protocol all contributing to the observed response. F639A mice, but not wild-type mice, showed enhanced motor activity following repeated

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

    Saurabh Singh


    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.

  6. 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

    吴作辉; 白文坤; 张吉臻; 张跃力; 申锷; 胡兵


    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.

  7. Bovine growth hormone-transgenic mice have major alterations in hepatic expression of metabolic genes.

    Olsson, Bob; Bohlooly-Y, Mohammad; Brusehed, Ola; Isaksson, Olle G P; Ahrén, Bo; Olofsson, Sven-Olof; Oscarsson, Jan; Törnell, Jan


    Transgenic mice overexpressing growth hormone (GH) have been extensively used to study the chronic effects of elevated serum levels of GH. GH is known to have many acute effects in the liver, but little is known about the chronic effects of GH overexpression on hepatic gene expression. Therefore, we used DNA microarray to compare gene expression in livers from bovine GH (bGH)-transgenic mice and littermates. Hepatic expression of peroxisome proliferator-activated receptor-alpha (PPARalpha) and genes involved in fatty acid activation, peroxisomal and mitochondrial beta-oxidation, and production of ketone bodies was decreased. In line with this expression profile, bGH-transgenic mice had a reduced ability to form ketone bodies in both the fed and fasted states. Although the bGH mice were hyperinsulinemic, the expression of sterol regulatory element-binding protein (SREBP)-1 and most lipogenic enzymes regulated by SREBP-1 was reduced, indicating that these mice are different from other insulin-resistant models with respect to expression of SREBP-1 and its downstream genes. This study also provides several candidate genes for the well-known association between elevated GH levels and cardiovascular disease, e.g., decreased expression of scavenger receptor class B type I, hepatic lipase, and serum paraoxonase and increased expression of serum amyloid A-3 protein. We conclude that bGH-transgenic mice display marked changes in hepatic genes coding for metabolic enzymes and suggest that GH directly or indirectly regulates many of these hepatic genes via decreased expression of PPARalpha and SREBP-1.

  8. Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

    Ikeda, Kazuhiro [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Tsukui, Tohru [Experimental Animal Laboratory, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Imazawa, Yukiko; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Inoue, Satoshi, E-mail: [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)


    Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

  9. Lymphocytes from wasted mice express enhanced spontaneous and {gamma}-ray-induced apoptosis

    Woloschak, G.E. [Argonne National Lab., IL (United States)]|[Loyola Univ. Medical Center, Maywood, IL (United States); Chang-Liu, Chin-Mei [Argonne National Lab., IL (United States); Chung, Jen; Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States)


    Mice bearing the autosomal recessive mutation wasted (wst/wst) display a disease pattern including faulty repair of DNA damage in lymphocytes after radiation exposure, neurologic abnormalities, and immunodeficiency. Many of the features of this mouse model have suggested a premature or increased spontaneous frequency of apoptosis in thymocytes; past work has shown an inability to establish cultured T cell lines, an abnormally high death rate of stimulated T cells in culture, and an increased sensitivity of T cells to the killing effects of ionizing radiations in wst/wst mice relative to controls. The experiments reported here were designed to examine splenic and thymic lymphocytes from wasted and control mice for signs of early apoptosis. Our results revealed enhanced expression of Rp-8 mRNA (associated with apoptosis) in thymic lymphocytes and reduced expression in splenic lymphocytes of wst/wst mice relative to controls; expression of Rp-2 and Td-30 mRNA (induced during apoptosis) were not detectable in spleen or thymus. Higher spontaneous DNA fragmentation was observed in wasted mice than in controls; however, {gamma}-ray-induced DNA fragmentation peaked at a lower dose and occurred to a greater extent in wasted mice relative to controls. These results provide evidence for high spontaneous and {gamma}-ray-induced apoptosis in T cells of wasted mice as a mechanism underlying the observed lymphocyte and DNA repair abnormalities.

  10. Microbiota prevents cholesterol loss from the body by regulating host gene expression in mice.

    Zhong, Chun-Yan; Sun, Wei-Wei; Ma, Yinyan; Zhu, Hongling; Yang, Pan; Wei, Hong; Zeng, Ben-Hua; Zhang, Qian; Liu, Yu; Li, Wen-Xia; Chen, Yixin; Yu, Liqing; Song, Zhi-Yuan


    We have previously observed that knockout of Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol transporter essential for intestinal cholesterol absorption, reduces the output of dry stool in mice. As the food intake remains unaltered in NPC1L1-knockout (L1-KO) mice, we hypothesized that NPC1L1 deficiency may alter the gut microbiome to reduce stool output. Consistently, here we demonstrate that the phyla of fecal microbiota differ substantially between L1-KO mice and their wild-type controls. Germ-free (GF) mice have reduced stool output. Inhibition of NPC1L1 by its inhibitor ezetimibe reduces stool output in specific pathogen-free (SPF), but not GF mice. In addition, we show that GF versus SPF mice have reduced intestinal absorption and increased fecal excretion of cholesterol, particularly after treatment with ezetimibe. This negative balance of cholesterol in GF mice is associated with reduced plasma and hepatic cholesterol, and likely caused by reduced expression of NPC1L1 and increased expression of ABCG5 and ABCG8 in small intestine. Expression levels of other genes in intestine and liver largely reflect a state of cholesterol depletion and a decrease in intestinal sensing of bile acids. Altogether, our findings reveal a broad role of microbiota in regulating whole-body cholesterol homeostasis and its response to a cholesterol-lowering drug, ezetimibe.

  11. Ectopic TLX1 expression accelerates malignancies in mice deficient in DNA-PK.

    Konstantin Krutikov

    Full Text Available The noncluster homeobox gene HOX11/TLX1 (TLX1 is detected at the breakpoint of the t(10;14(q24;q11 chromosome translocation in patients with T cell acute lymphoblastic leukemia (T-ALL. This translocation results in the inappropriate expression of TLX1 in T cells. The oncogenic potential of TLX1 was demonstrated in IgHμ-TLX1(Tg mice which develop mature B cell lymphoma after a long latency period, suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed IgHμ-TLX1(Tg mice with mice deficient in the DNA repair enzyme DNA-PK (Prkdc(Scid/Scid mice. IgHµ-TLX1(TgPrkdc(Scid/Scid mice developed T-ALL and acute myeloid leukemia (AML with reduced latency relative to control Prkdc(Scid/Scid mice. Further analysis of thymi from premalignant mice revealed greater thymic cellularity concomitant with increased thymocyte proliferation and decreased apoptotic index. Moreover, premalignant and malignant thymocytes exhibited impaired spindle checkpoint function, in association with aneuploid karyotypes. Gene expression profiling of premalignant IgHµ-TLX1(TgPrkdc(Scid/Scid thymocytes revealed dysregulated expression of cell cycle, apoptotic and mitotic spindle checkpoint genes in double negative 2 (DN2 and DN3 stage thymocytes. Collectively, these findings reveal a novel synergy between TLX1 and impaired DNA repair pathway in leukemogenesis.

  12. Expression of monocyte chemoattractant protein-1 in the pancreas of mice

    LI Dong; ZHU Su-wen; LIU Dong-juan; LIU Guo-liang; SHAN Zhong-yan


    Background Type 1 diabetes has been recognized as an organ specific autoimmune disease owing to the immune destruction of pancreatic islet β cells in genetically susceptible individuals.In both human and rodent models of type 1 diabetes, such as nonobese diabetic (NOD) mice, biobreeding rats, the disease has a distinct stage characterized by immune cells infiltrating in the pancreas (insulitis).The major populations of infiltrating cells are macrophages and T lymphocytes.Therefore, immune cell infiltration of pancreatic islets may be a crucial step in the pathogenesis of type 1 diabetes.Monocyte chemoattractant protein-1 can specifically attract monocytes in vivo.Interferon induced protein-10 has chemoattractant effects on the activated lymphocytes.In this study, we analysed the expression of monocyte chemoattractant protein-1 in the pancreas of mice and interferon inducible protein-10 mRNA in the pancreas of NOD mice, and discussed their possible role in the pathogenesis of type 1 diabetes.Methods The immunohistochemical method and immunoelectronmicroscopy were used to evaluate the expression of monocyte chemoattractant protein-1 in the pancreas of NOD mice and BALB/c mice.RT-PCR was used to evaluate the expression of monocyte chemoattractant protein-1 and interferon inducible protein mRNA in NOD mice.Results Monocyte chemoattractant protein-1 was positive in the pancreas of NOD mice, whereas negative in the pancreas of BALB/C mice.RT-PCR showed that monocyte chemoattractant protein-1 and interferon inducible protein-10 mRNA could be found in the pancreas of NOD mice.Immunoelectronmicroscopy demonstrated that monocyte chemoattractant protein-1 was produced by β cells and stored in the cytoplasm of the cells.Conclusions Pancreatic islet β cells produce monocyte chemoattractantprotein-1 in NOD mice.Monocyte chemoattractant protein-1 may play an important part in the pathogenesis of type 1 diabetes by attracting monocytes/macrophages to infiltrate pancreatic

  13. RNA expression profiling in brains of familial hemiplegic migraine type 1 knock-in mice.

    de Vries, Boukje; Eising, Else; Broos, Ludo A M; Koelewijn, Stephany C; Todorov, Boyan; Frants, Rune R; Boer, Judith M; Ferrari, Michel D; Hoen, Peter A C 't; van den Maagdenberg, Arn M J M


    Various CACNA1A missense mutations cause familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of migraine with aura. FHM1 mutation R192Q is associated with pure hemiplegic migraine, whereas the S218L mutation causes hemiplegic migraine, cerebellar ataxia, seizures, and mild head trauma-induced brain edema. Transgenic knock-in (KI) migraine mouse models were generated that carried either the FHM1 R192Q or the S218L mutation and were shown to exhibit increased CaV2.1 channel activity. Here we investigated their cerebellar and caudal cortical transcriptome. Caudal cortical and cerebellar RNA expression profiles from mutant and wild-type mice were studied using microarrays. Respective brain regions were selected based on their relevance to migraine aura and ataxia. Relevant expression changes were further investigated at RNA and protein level by quantitative polymerase chain reaction (qPCR) and/or immunohistochemistry, respectively. Expression differences in the cerebellum were most pronounced in S218L mice. Particularly, tyrosine hydroxylase, a marker of delayed cerebellar maturation, appeared strongly upregulated in S218L cerebella. In contrast, only minimal expression differences were observed in the caudal cortex of either mutant mice strain. Despite pronounced consequences of migraine gene mutations at the neurobiological level, changes in cortical RNA expression in FHM1 migraine mice compared to wild-type are modest. In contrast, pronounced RNA expression changes are seen in the cerebellum of S218L mice and may explain their cerebellar ataxia phenotype.

  14. Nursing frequency alters circadian patterns of mammary gene expression in lactating mice

    Milking frequency impacts lactation in dairy cattle and in rodent models of lactation. The role of circadian gene expression in this process is unknown. The hypothesis tested was that changing nursing frequency alters the circadian patterns of mammary gene expression. Mid-lactation CD1 mice were stu...

  15. Regulation of human clotting factor IX cDNA expression in transgenic mice

    胡以平; 邱信芳; 薛京伦; 刘祖洞


    To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

  16. Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

    D Feder; Rugollini, M.; Santomauro Jr,A.; de Oliveira, L. P.; Lioi,V.P.; R. dos Santos; Ferreira, L.G.; Nunes,M.T.; M.H. Carvalho; P.O. Delgado; A.A.S. Carvalho; Fonseca, F.L.A.


    Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. in this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength ...

  17. Effect of telmisartan on expression of protein kinase C-α in kidneys of diabetic mice

    Li-jun YAO; Jian-qing WANG; Hong ZHAO; Jian-she LIU; An-guo DENG


    Aim: To investigate the effects of angiotensin receptor blocker (ARB) telmisartan on the expression and distribution of protein kinase C (PKC)-α in the kidneys of diabetic mice.Methods: Diabetic mice were induced with streptozotocin and a group of them were randomly selected for treatment with telmisartan. After 6 weeks, the expression and localization of PKC-α in the renal cortex, and the outer and inner medulla were assessed by immunohistochemistry and semiquantitative Western blotting. In addition, expressions of PKC-α, transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor (VEGF) in glomemli were measured by semiquantitative immunohistochemistry.Results: Diabetic and nor-mal mice showed similar distributions of PKC-α in the kidneys. The expression of PKC-α was found in glomeruli, epithelial cells of proximal tubules, and medullary-collecting duct, while not in the medullary and cortical thick ascending limb, and was different in the epithelial cells of proximal tubules of diabetic nephropathy (DN) mice, PKC-α was mostly translocated from the basement membrane to the apical membrane, whereas it was largely translocated from the apical membrane to the basement membrane in epithelial cells of the inner medullary-collecting duct.Western blotting detected increased expression of PKC-α in the renal cortex and outer medulla, but not in the inner medulla of DN mice. Enhanced expressions of PKC-α TGF-β, and VEGF were shown in the glomeruli of DN mice, where PKC-α exhibited a correlation to VEGF, but no correlation to TGF-β1. ARB telmisartan attenuated alterations of PKC-α as mentioned earlier in the DN mice.Conclusion: Our findings suggest that PKC-α may play a role in the pathogenesis of DN, and that the nephroprotective effects of ARB telmisartan may be partly associated with its influence on PKC-α.

  18. [Expression of various matrix metalloproteinases in mice with hyperoxia-induced acute lung injury].

    Zhang, Xiang-feng; Ding, Shao-fang; Gao, Yuan-ming; Liang, Ying; Foda, Hussein D


    To investigate the role of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) in the pathogenesis of acute lung injury induced by hyperoxia. Fifty four mice were exposed in sealed cages to >98% oxygen (for 24-72 hours), and another 18 mice to room air. The severity of lung injury was assessed, and the expression of mRNA and protein of MMP-2, MMP-9 and EMMPRIN in lung tissue, after exposure for 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Hyperoxia caused acute lung injury; this was accompanied by increased expression of an upregulation of MMP-2, MMP-9 and EMMPRIN mRNA and protein in lung tissues. Hyperoxia causes acute lung injury in mice; increases in MMP-2, MMP-9 and EMMPRIN may play an important role in the development of hyperoxia induced lung injury in mice.

  19. Differential expression of metallothioneins in the CNS of mice with experimental autoimmune encephalomyelitis

    Espejo, C; Carrasco, J; Hidalgo, J


    , and to a lower extent in the brain. Interferon-gamma receptor knockout mice suffered from a more severe experimental autoimmune encephalomyelitis, and interestingly showed a higher metallothioneins-I+II induction in both white and grey matter of the spinal cord and in the brain. In contrast...... during experimental autoimmune encephalomyelitis in interferon-gamma receptor knockout mice with two different genetic backgrounds: 129/Sv and C57BL/6x129/Sv.Mice with experimental autoimmune encephalomyelitis showed a significant induction of metallothioneins-I+II in the spinal cord white matter...... to the metallothioneins-I+II isoforms, metallothionein-III expression remained essentially unaltered during experimental autoimmune encephalomyelitis; interferon-gamma receptor knockout mice showed an altered metallothionein-III expression (a slight increase in the spinal cord white matter) only in the C57BL/6x129/Sv...

  20. A humanized pattern of aromatase expression is associated with mammary hyperplasia in mice.

    Zhao, Hong; Pearson, Elizabeth K; Brooks, David C; Coon, John S; Chen, Dong; Demura, Masashi; Zhang, Ming; Clevenger, Charles V; Xu, Xia; Veenstra, Timothy D; Chatterton, Robert T; DeMayo, Francesco J; Bulun, Serdar E


    Aromatase is essential for estrogen production and is the target of aromatase inhibitors, the most effective endocrine treatment for postmenopausal breast cancer. Peripheral tissues in women, including the breast, express aromatase via alternative promoters. Female mice lack the promoters that drive aromatase expression in peripheral tissues; thus, we generated a transgenic humanized aromatase (Arom(hum)) mouse line containing a single copy of the human aromatase gene to study the link between aromatase expression in mammary adipose tissue and breast pathology. Arom(hum) mice expressed human aromatase, driven by the proximal human promoters II and I.3 and the distal promoter I.4, in breast adipose fibroblasts and myoepithelial cells. Estrogen levels in the breast tissue of Arom(hum) mice were higher than in wild-type mice, whereas circulating levels were similar. Arom(hum) mice exhibited accelerated mammary duct elongation at puberty and an increased incidence of lobuloalveolar breast hyperplasia associated with increased signal transducer and activator of transcription-5 phosphorylation at 24 and 64 wk. Hyperplastic epithelial cells showed remarkably increased proliferative activity. Thus, we demonstrated that the human aromatase gene can be expressed via its native promoters in a wide variety of mouse tissues and in a distribution pattern nearly identical to that of humans. Locally increased tissue levels, but not circulating levels, of estrogen appeared to exert hyperplastic effects on the mammary gland. This novel mouse model will be valuable for developing tissue-specific aromatase inhibition strategies.

  1. Dendrobium nobile Lindl. alkaloids regulate metabolism gene expression in livers of mice.

    Xu, Yun-Yan; Xu, Ya-Sha; Wang, Yuan; Wu, Qin; Lu, Yuan-Fu; Liu, Jie; Shi, Jing-Shan


    In our previous studies, Dendrobium nobile Lindl. alkaloids (DNLA) has been shown to have glucose-lowering and antihyperlipidaemia effects in diabetic rats, in rats fed with high-fat diets, and in mice challenged with adrenaline. This study aimed to examine the effects of DNLA on the expression of glucose and lipid metabolism genes in livers of mice. Mice were given DNLA at doses of 10-80 mg/kg, po for 8 days, and livers were removed for total RNA and protein isolation to perform real-time RT-PCR and Western blot analysis. Dendrobium nobile Lindl. alkaloids increased PGC1α at mRNA and protein levels and increased glucose metabolism gene Glut2 and FoxO1 expression. DNLA also increased the expression of fatty acid β-oxidation genes Acox1 and Cpt1a. The lipid synthesis regulator Srebp1 (sterol regulatory element-binding protein-1) was decreased, while the lipolysis gene ATGL was increased. Interestingly, DNLA increased the expression of antioxidant gene metallothionein-1 and NADPH quinone oxidoreductase-1 (Nqo1) in livers of mice. Western blot on selected proteins confirmed these changes including the increased expression of GLUT4 and PPARα. DNLA has beneficial effects on liver glucose and lipid metabolism gene expressions, and enhances the Nrf2-antioxidant pathway gene expressions, which could play integrated roles in regulating metabolic disorders. © 2017 Royal Pharmaceutical Society.

  2. Dual mechanisms of rapid expression of anxiety-related behavior in pilocarpine-treated epileptic mice.

    Otsuka, Shintaro; Ohkido, Taro; Itakura, Makoto; Watanabe, Shigeru; Yamamori, Saori; Iida, Yuuki; Saito, Masanori; Miyaoka, Hitoshi; Takahashi, Masami


    A mouse model of epilepsy was generated by inducing status epilepticus (SE) for either 1.5 or 4.5h with pilocarpine to study anxiety-related behaviors, changes in the electroencephalogram of the cerebral cortex and hippocampus, and expression of hippocampal proteins. The viability and rate of success of SE induction were high in C57BL/6N mice but not in C57BL/6J mice. C57BL/6N mice were immotile during the first 2days after SE; however, by the third day, most mice were recovered and exhibited strong anxiety-related behaviors in response to the light/dark preference test and open field test. There was a striking difference in the temporal appearance of anxiety-related behavior between the two SE durations: 1.5h SE mice exhibited strong anxiety-related behavior 3days after SE that gradually attenuated over the next few weeks, whereas 4.5h SE mice exhibited strong anxiety-related behavior 3days after SE that persisted even at nearly 1year after SE. Mice receiving both SE durations exhibited generalized seizures (GS) after SE; however, there was a marked difference in the timing and duration of GS appearance. Mice in the 4.5h SE group exhibited spontaneous GS from 4days to at least 96days after SE. In contrast, mice in the 1.5h SE group exhibited GS only within the first several days after SE; however, epileptic spike clusters continuously appeared in the cerebral cortex and hippocampus for up to twelve days after SE. Among the hippocampal proteins tested, only brain derived-neurotrophic factor (BDNF) exhibited altered expression in parallel with anxiety-related behavior. These results showed the possibility that BDNF expression in the hippocampus might cause anxiety-related behavior in adulthood.

  3. Effect of Yiguanjian decoction on cell differentiation and proliferation in CCl4-treated mice

    Xiao-Ling Wang; Dong-Wei Jia; Hui-Yang Liu; Xiao-Feng Yan; Ting-Jie Ye; Xu-Dong Hu; Bo-Qin Li


    AIM:To investigate the cellular mechanisms of action of Yiguanjian (YGJ) decoction in treatment of chronic hepatic injury.METHODS:One group of mice was irradiated,and received enhanced green fluorescent protein (EGFP)-positive bone marrow transplants followed by 13 wk of CCl4 injection and 6 wk of oral YGJ administration.A second group of Institute for Cancer Research mice was treated with 13 wk of CCl4 injection and 6 wk of oral YGJ administration.Liver function,histological changes in the liver,and Hyp content were analyzed.The expression of α-smooth muscle actin (α-SMA),F4/80,albumin (Alb),EGFP,mitogen-activated protein kinase-2 (PKM2),Ki-67,α fetoprotein (AFP),monocyte chemotaxis protein-1 and CC chemokine receptor 2 were assayed.RESULTS:As hepatic damage progressed,EGFP-positive marrow cells migrated into the liver and were mainly distributed along the fibrous septa.They showed a conspicuous coexpression of EGFP with α-SMA and F4/80 but no coexpression with AIb.Moreover,the expression of PKM2,AFP and Ki-67 was enhanced dynamically and steadily over the course of liver injury.YGJ abrogated the increases in the number of bone marrow-derived fibrogenic cells in the liver,inhibited expression of both progenitor and mature hepatocyte markers,and reduced fibrogenesis.CONCLUSION:YGJ decoction improves liver fibrosis by inhibiting the migration of bone marrow cells into the liver as well as inhibiting their differentiation and suppressing the proliferation of both progenitors and hepatocytes in the injured liver.

  4. Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

    Maria Arantzazu Sanchez-Fernandez

    Full Text Available BACKGROUND: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. METHODOLOGY/PRINCIPAL FINDING: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK, a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/-LacZ+/- adult mice show a Cre-dependent β-galactosidase activity after tamoxifen stimulation. CONCLUSIONS/SIGNIFICANCE: We have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

  5. Decreasing TfR1 expression reverses anemia and hepcidin suppression in β-thalassemic mice.

    Li, Huihui; Choesang, Tenzin; Bao, Weili; Chen, Huiyong; Feola, Maria; Garcia-Santos, Daniel; Li, Jie; Sun, Shuming; Follenzi, Antonia; Pham, Petra; Liu, Jing; Zhang, Jinghua; Ponka, Prem; An, Xiuli; Mohandas, Narla; Fleming, Robert; Rivella, Stefano; Li, Guiyuan; Ginzburg, Yelena


    Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apo-transferrin leads to more effective erythropoiesis, decreasing erythroferrone and de-repressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apo-transferrin's effect is mediated via decreased TfR1 expression, and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apo-transferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect which can be reversed by the administration of exogenous apo-transferrin. In vitro experiments demonstrate that apo-transferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1+/- mice--themselves exhibiting iron-restricted erythropoiesis with increased hepcidin--with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin de-repression, and increased erythroid enucleation relative to β-thalassemic mice. Our data demonstrates for the first time that TfR1+/- haplo-insufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either via directly induced haplo-insufficiency or exogenous apo-transferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron

  6. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.

    Robert A Koza


    Full Text Available High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.

  7. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.

    Koza, Robert A; Nikonova, Larissa; Hogan, Jessica; Rim, Jong-Seop; Mendoza, Tamra; Faulk, Christopher; Skaf, Jihad; Kozak, Leslie P


    High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.

  8. Deletion of IL-18 Expression Ameliorates Spontaneous Kidney Failure in MRLlpr Mice.

    Bastian Schirmer

    Full Text Available The role of IL-18 in the pathogenesis of systemic lupus erythematosus is still not definitively solved. In this study, we generated MRLlpr mice, which develop a disease resembling systemic lupus erythematosus, genetically devoid of IL-18 expression. These mice in comparison to IL-18-competent MRLlpr mice show reduced signs of renal pathogenesis, while other parameters such as mean survival time, lymphadenopathy, constitutive interferon-γ production, and frequency of CD3+B220+ abnormal T cells were without differences. We conclude that in the systemic lupus erythematosus syndrom IL-18 is involved specifically in the renal pathogenesis.

  9. Serum miRNA expression profiles change in autoimmune vitiligo in mice.

    Shi, Yu-Ling; Weiland, Matthew; Lim, Henry W; Mi, Qing-Sheng; Zhou, Li


    It is widely believed that non-segmental vitiligo results from the autoimmune destruction of melanocytes. MicroRNAs (miRNAs), a class of small non-coding RNAs that negatively regulate gene expression, are involved in the immune cell development and function and regulate the development of autoimmune diseases. Recent studies demonstrate that functional miRNAs can be detected in the serum and serve as biomarkers of various diseases. In the present study, we used a mouse autoimmune vitiligo model, in which melanocyte autoreactive CD4+ T cells were adoptively transferred into Rag1(-/-) host mice. Serum miRNA expression was profiled in vitiligo developed mice and control mice using TaqMan RT-PCR arrays. We have found that the expressions of 20 serum miRNAs were changed in vitiligo mice compared to control mice. Three increased miRNAs, miR-146a, miR-191, and miR-342-3p, were further confirmed by a single TaqMan RT-PCR. Our findings suggest that miRNAs may be involved in vitiligo development and serum miRNAs could serve as serum biomarkers for vitiligo in mice.

  10. Ezrin is highly expressed in early thymocytes, but dispensable for T cell development in mice.

    Meredith H Shaffer

    Full Text Available BACKGROUND: Ezrin/radixin/moesin (ERM proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/- mice likely arise as a consequence of nutritional stress. CONCLUSIONS/SIGNIFICANCE: We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.

  11. Mice with early retinal degeneration show differences in neuropeptide expression in the suprachiasmatic nucleus

    Brown R Lane


    Full Text Available Abstract Background In mammals, the brain clock responsible for generating circadian rhythms is located in the suprachiasmatic nucleus (SCN of the hypothalamus. Light entrainment of the clock occurs through intrinsically photosensitive retinal ganglion cells (ipRGCs whose axons project to the SCN via the retinohypothalamic tract. Although ipRGCs are sufficient for photoentrainment, rod and cone photoreceptors also contribute. Adult CBA/J mice, which exhibit loss of rod and cone photoreceptors during early postnatal development, have greater numbers of ipRGCs compared to CBA/N control mice. A greater number of photosensitive cells might argue for enhanced light responses, however, these mice exhibit attenuated phase shifting behaviors. To reconcile these findings, we looked for potential differences in SCN neurons of CBA/J mice that might underly the altered circadian behaviors. We hypothesized that CBA/J mice have differences in the expression of neuropeptides in the SCN, where ipRGCs synapse. The neuropeptides vasoactive intestinal peptide (VIP and vasopressin (VP are expressed by many SCN neurons and play an important role in the generation of circadian rhythms and photic entrainment. Methods Using immunohistochemistry, we looked for differences in the expression of VIP and VP in the SCN of CBA/J mice, and using a light-induced FOS assay, we also examined the degree of retinal innervation of the SCN by ipRGCs. Results Our data demonstrate greater numbers of VIP-and VP-positive cells in the SCN of CBA/J mice and a greater degree of light-induced FOS expression. Conclusions These results implicate changes in neuropeptide expression in the SCN which may underlie the altered circadian responses to light in these animals.

  12. Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens

    Near, Richard I.; Smith, Richard S.; Toselli, Paul A.; Freddo, Thomas F.; Bloom, Alexander B.; Vanden Borre, Pierre; Seldin, David C.


    Purpose AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism. Methods AND-34−/− mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues. Results Western analyses confirmed total loss of expression in AND-34−/− splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34−/− mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34−/− mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34−/− mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34+/+ lens epithelial cells, it was markedly reduced in the AND-34−/− lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34+/+ than in AND-34−/− lens epithelium. Conclusions These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens. PMID:19365570

  13. Gene expression analyses implicate an alternative splicing program in regulating contractile gene expression and serum response factor activity in mice.

    Twishasri Dasgupta

    Full Text Available Members of the CUG-BP, Elav-like family (CELF regulate alternative splicing in the heart. In MHC-CELFΔ transgenic mice, CELF splicing activity is inhibited postnatally in heart muscle via expression of a nuclear dominant negative CELF protein under an α-myosin heavy chain promoter. MHC-CELFΔ mice develop dilated cardiomyopathy characterized by alternative splicing defects, enlarged hearts, and severe contractile dysfunction. In this study, gene expression profiles in the hearts of wild type, high- and low-expressing lines of MHC-CELFΔ mice were compared using microarrays. Gene ontology and pathway analyses identified contraction and calcium signaling as the most affected processes. Network analysis revealed that the serum response factor (SRF network is highly affected. Downstream targets of SRF were up-regulated in MHC-CELFΔ mice compared to the wild type, suggesting an increase in SRF activity. Although SRF levels remained unchanged, known inhibitors of SRF activity were down-regulated. Conversely, we found that these inhibitors are up-regulated and downstream SRF targets are down-regulated in the hearts of MCKCUG-BP1 mice, which mildly over-express CELF1 in heart and skeletal muscle. This suggests that changes in SRF activity are a consequence of changes in CELF-mediated regulation rather than a secondary result of compensatory pathways in heart failure. In MHC-CELFΔ males, where the phenotype is only partially penetrant, both alternative splicing changes and down-regulation of inhibitors of SRF correlate with the development of cardiomyopathy. Together, these results strongly support a role for CELF-mediated alternative splicing in the regulation of contractile gene expression, achieved in part through modulating the activity of SRF, a key cardiac transcription factor.

  14. Renal NHE expression and activity in neonatal NHE3- and NHE8-null mice.

    Pirojsakul, Kwanchai; Gattineni, Jyothsna; Dwarakanath, Vangipuram; Baum, Michel


    Na(+)/H(+) exchanger (NHE)3 is the predominant NHE on the brush-border membrane of the proximal tubule in adult animals. NHE8 has been localized to the brush-border membrane of proximal tubules and is more highly expressed in neonates than in adult animals. However, the relative role of NHE8 in neonatal renal acidification is unclear. The present study examined if there was a compensatory increase in NHE3 in NHE8-null neonatal mice and whether there was a compensatory increase in NHE8 in NHE3-null neonatal mice. In addition, we examined whether wild-type, NHE3-null, and NHE8-null mice had an increase in NHE activity in response to metabolic acidosis. We found that at baseline, there was comparable renal NHE3 mRNA, total protein, and brush-border membrane protein abundance as in neonatal control and NHE8-null mice. There was comparable renal NHE8 mRNA, total protein, and brush-border membrane protein abundance in NHE3-null neonatal and control mice. Both NHE3- and NHE8-null mice had a comparable but lower rate of NHE activity than control mice. We next imposed metabolic acidosis in wild-type, NHE3-null, and NHE8-null mice. Acidemic NHE8-null mice had an increase in brush-border membrane vesicle NHE3 protein abundance and NHE activity compared with vehicle-treated mice. Likewise, NHE3-null mice had an increase in NHE8 brush-border membrane protein abundance and NHE activity in response to metabolic acidosis. In conclusion, both NHE3 and NHE8 likely play a role in neonatal acidification.

  15. Reduced expression of the ATRX gene, a chromatin-remodeling factor, causes hippocampal dysfunction in mice.

    Nogami, Tatsuya; Beppu, Hideyuki; Tokoro, Takashi; Moriguchi, Shigeki; Shioda, Norifumi; Fukunaga, Kohji; Ohtsuka, Toshihisa; Ishii, Yoko; Sasahara, Masakiyo; Shimada, Yutaka; Nishijo, Hisao; Li, En; Kitajima, Isao


    Mutations of the ATRX gene, which encodes an ATP-dependent chromatin-remodeling factor, were identified in patients with α-thalassemia X-linked mental retardation (ATR-X) syndrome. There is a milder variant of ATR-X syndrome caused by mutations in the Exon 2 of the gene. To examine the impact of the Exon 2 mutation on neuronal development, we generated ATRX mutant (ATRX(ΔE2)) mice. Truncated ATRX protein was produced from the ATRX(ΔE2) mutant allele with reduced expression level. The ATRX(ΔE2) mice survived and reproduced normally. There was no significant difference in Morris water maze test between wild-type and ATRX(ΔE2) mice. In a contextual fear conditioning test, however, total freezing time was decreased in ATRX(ΔE2) mice compared to wild-type mice, suggesting that ATRX(ΔE2) mice have impaired contextual fear memory. ATRX(ΔE2) mice showed significantly reduced long-term potentiation in the hippocampal CA1 region evoked by high-frequency stimulation. Moreover, autophosphorylation of calcium-calmodulin-dependent kinase II (αCaMKII) and phosphorylation of glutamate receptor, ionotropic, AMPA 1 (GluR1) were decreased in the hippocampi of the ATRX(ΔE2) mice compared to wild-type mice. These findings suggest that ATRX(ΔE2) mice may have fear-associated learning impairment with the dysfunction of αCaMKII and GluR1. The ATRX(ΔE2) mice would be useful tools to investigate the role of the chromatin-remodeling factor in the pathogenesis of abnormal behaviors and learning impairment.

  16. Small animal PET imaging of HSV1-tk gene expression with {sup 124}IVDU in liver by the hydrodynamic injection

    Song, I. H.; Lee, T. S.; Woo, S. G.; Jeong, J. H.; Kang, J. H.; Kim, K. M.; Chun, K. J.; Choi, C. W.; Lim, S. M. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)


    The liver is an important target organ for gene transfer due to its capacity for synthesizing serum protein and its involvement in numerous genetic diseases. High level of foreign gene expression in liver can be achieved by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), so called hydrodynamic injection. This study is aimed to evaluate liver specific-gene expression of herpes simplex virus type 1 thymidine kinase(HSV1-tk) by hydrodynamic injection and image HSV1-tk expression using {sup 124}IVDU-PET. We constructed herpes simplex virus type 1 thymidine kinase (HSV1-tk)-expressing pDNA (pHSV1-tk) modified from pEGFP-N1. Hydrodynamic injection was performed using 40 {mu}g of plasmid (pEGFP/N1 or pHSV1-tk) in 2 ml of 0.85% saline solution for 20{approx}22g mice in 5 seconds intravenously. At 1 d post-hydrodynamic injection, biodistribution study was performed at 2 h post-injection of radiolabeled IVDU, fluorescence image was obtained using optical imager and small animal PET image was acquired with {sup 124}IVDU at 2 h post-injection. After PET imaging, digital whole body autoradiography (DWBA) was performed. Expression of HSV1-tk and EGFP was confirmed by RT-PCR in each liver tissue. In liver of pHSV1-tk and pEGFP/N1 injection groups, {sup 123}IVDU uptake was 5.65%ID/g and 0.98%ID/g, respectively. {sup 123}IVDU uptake in liver of pHSV1-tk injection group showed 5.7-fold higher than that of pEGFP/N1 injection group (p<0.01). On the other hand, the liver of pEGFP/N1 injection group showed fluorescence activity. In small animal PET images, {sup 124}IVDU uptake was selectively localized in liver of pHSV1-tk injection group and also checked in DWBA, but showed minimal uptake in liver of pEGFP/N1 injection mice. Hydrodynamic injection was effective to liver-specific delivery of plasmid DNA. Small animal PET image of {sup 124}IVDU could be used in the evaluation of noninvasive reporter gene imaging in liver.

  17. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    Fromont-Racine, M; Bucchini, D; Madsen, O


    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...... of the transgene was observed in cell types other than beta-islet cells....

  18. Time course degeneration and expression of glial fibrillary acidic protein in mer-knockout mice

    LIANG Xiao-ying; WANG Huai-zhou; WANG Ning-li


    Background Muller cells in the mammalian retina normally express low levels of glial fibrillary acidic protein (GFAP); however, its expression is upregulated in response to the loss of retinal neurons. The change in expression of GFAP is one of the earliest indicators of retinal damage and is correlated with the time course of disease. The aim of this study was to investigate the time course of degeneration and the expression of GFAP in the retina of mer knockout mice. Methods A total of 30 mer knockout mice, aged from 15-20 days to 1 year and 32 age-matched wild type mice as controls were tested. Immunohistochemistry was used to show the expression of GFAP in the central and peripheral retina of mer knockout and control mice at postnatal age of 15 days (P15d), 20 days (P20d), 4 weeks (P4w), 6 weeks (P6w), 8 weeks (P8w), 3 months (P3m), 6 months (P6m) and 1 years (P1y).Results The expression of GFAP in the central and peripheral retina of wild type mice was limited to the retinal ganglion cell and nerve fiber layers. In the central retina of mer knockout mice, GFAP expression was upregulated at P4w and GFAP immunolabelling penetrates across the entire thickness of the retina at P8w; whereas in the peripheral retina, the GFAP expression was upregulated at P20d and GFAP immunolabelling penetrates the entire retina after P4w. Conclusions Increased expression of GFAP in Muller cells of mer knockout mice occur at P20d in the peripheral retina and P4w in the central retina. GFAP expression in Muller cells appears to be a secondary response to the loss of retinal neurons. Increased expression of GFAP may occur prior to any detectable morphological changes in the retina. This study suggests that the loss of retinal neurons may begin in the early stages of retinitis pigmentosa, prior to the discovery of any morphological changes in the retina.

  19. Striatal adenosine signaling regulates EAAT2 and astrocytic AQP4 expression and alcohol drinking in mice.

    Lee, Moonnoh R; Ruby, Christina L; Hinton, David J; Choi, Sun; Adams, Chelsea A; Young Kang, Na; Choi, Doo-Sup


    Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.

  20. Adipose tissue gene expression profiles in ob/ob mice treated with leptin.

    Zhang, Wei; Della-Fera, Mary Anne; Hartzell, Diane L; Hausman, Dorothy; Baile, Clifton A


    Leptin plays a critical role in regulating body weight, lipid metabolism, apoptosis and microvasculature of adipose tissue. To explore multiple signaling pathways of leptin action on adipose tissue, real-time PCR utilizing TaqMan low-density arrays was performed to compare mRNA expression in adipose tissue of ob/ob mice treated with vehicle or leptin (2.5 microg/d or 10 microg/d) for 14 days via subcutaneous osmotic minipumps. Of the 24 target genes selected for characterization, many were differentially expressed between control ob/ob mice and leptin-treated ob/ob mice. Increases in mRNA expression were found for hormone sensitive lipase (HSL), uncoupling protein 2 (UCP2), adrenergic receptor 3 (ADR3), mitofusin 2 (Mfn2), sirtuin 3 (Sirt3), transcription factor sterol regulatory element binding factor 1 (SREBF1), Bcl-2, Bax, Caspase 3, tumor necrosis factor alpha (TNFalpha), adiponectin and angiopoietin 2 (Ang-2). Decreases in expression were found for stearoyl-coenzyme A desaturase 1 (SCD1), fatty acid synthase (FAS), and retinol binding protein 4 (RBP4). There were no changes in expression of transcription factors involved in adipocyte differentiation (C/EBPalpha, PPARalpha, and PPARgamma). These results confirm that alterations in the expression of specific adipose tissue genes including those associated with the promotion of lipid mobilization, energy dissipation, and apoptosis may mediate leptin-induced fat loss in ob/ob mice.

  1. Effects of over-expressing resistin on glucose and lipid metabolism in mice

    You LIU; Qun WANG; Ying-bin PAN; Zhi-jie GAO; Yan-fen LIU; Shao-hong CHEN


    Resistin, a newly discovered peptide hormone mainly secreted by adipose tissues, is present at high levels in serum of obese mice and may be a potential link between obesity and insulin resistance in rodents. However, some studies of rat and mouse models have associated insulin resistance and obesity with decreased resistin expression. In humans, no relationship between resistin level and insulin resistance or adiposity was observed. This suggests that additional studies are necessary to determine the specific role of resistin in the regulation of energy metabolism and adipogenesis. In the present study, we investigated the effect of resistin in vivo on glucose and lipid metabolism by over-expressing resistin in mice by intramuscular injection of a recombinant eukaryotic expression vector pcDNA3.1-Retn encoding porcine resistin gene. After injection, serum resistin and serum glucose (GLU) levels were significantly increased in the pcDNA3.1-Retn-treated mice; there was an obvious difference in total cholesterol (TC) level between the experiment and the control groups on Day 30. In pcDNA3.1-Retn-treated mice, both free fatty acid (FFA) and high density lipoprotein (HDL) cholesterol levels were markedly lower than those of control, whereas HDL cholesterol and triglyceride (TG) levels did not differ between the two groups. Furthermore, lipase activity was expressly lower on Day 20. Our data suggest that resistin over-expressed in mice might be responsible for insulin resistance and parameters related to glucose and lipid metabolism were changed accordingly.

  2. Olfactory ensheathing cells of hamsters, rabbits, monkeys, and mice express α-smooth muscle actin.

    Rawji, Khalil S; Zhang, Shannon X; Tsai, Ying-Yu; Smithson, Laura J; Kawaja, Michael D


    Olfactory ensheathing cells (OECs) are the chief glial population of the mammalian olfactory nervous system, residing in the olfactory mucosa and at the surface of the olfactory bulb. We investigated the neurochemical features of OECs in a variety of mammalian species (including adult hamsters, rabbits, monkeys, and mice, as well as fetal pigs) using three biomarkers: α-smooth muscle actin (αSMA), S100β, and glial fibrillary acidic protein (GFAP). Mucosal and bulbar OECs from all five mammalian species express S100β. Both mucosal and bulbar OECs of monkeys express αSMA, yet only bulbar OECs of hamsters and only mucosal OECs of rabbits express αSMA as well. Mucosal OECs, but not bulbar OECs, also express GFAP in hamsters and monkeys; mice, by comparison, have only a sparse population of OECs expressing GFAP. Though αSMA immunostaining is not detected in OECs of adult mice, GFAP-expressing mucosal OECs isolated from adult mice do coexpress αSMA in vitro. Moreover, mucosal OECs from adult mutant mice lacking αSMA expression display perturbed cellular morphology (i.e., fewer cytoplasmic processes extending among the hundreds of olfactory axons in the olfactory nerve fascicles and nuclei having degenerative features). In sum, these findings highlight the efficacy of αSMA and S100β as biomarkers of OECs from a variety of mammalian species. These observations provide definitive evidence that mammalian OECs express the structural protein αSMA (at various levels of detection), which appears to play a pivotal role in their ensheathment of olfactory axons.

  3. Mice that are fed a high-fat diet display increased hepcidin expression in adipose tissue.

    Gotardo, Érica Martins Ferreira; dos Santos, Aline Noronha; Miyashiro, Renan Akira; Gambero, Sheley; Rocha, Thalita; Ribeiro, Marcelo Lima; Gambero, Alessandra


    Since the discovery that hepcidin is expressed in the adipose tissue of obese subjects, attention has been increasingly focused on alterations in iron homeostasis that are associated with adiposity. We examined the production of hepcidin, the expression of hepcidin-related genes and the iron content of the adipose tissue in obesity using Swiss mice fed a high-fat diet (HFD). The mice were maintained on a control diet or HFD for 12 or 24 wk, and body weight, adiposity and glucose homeostasis were evaluated. The expression of several genes (hepcidin, TfR1, TfR2, DMT1, FT-heavy, ferroportin, IRP-1, IRP-2 and HIF-1) and the protein expression of hepcidin and IL-6 were quantified. The iron level was assessed using a Prussian blue reaction in paraffin-embedded tissue. After 24 wk on the HFD, we observed increases in the levels of hepcidin in the serum and the visceral adipose tissue. The IL-6 levels also increased in the visceral adipose tissue. Adipocytes isolated from the visceral adipose tissues of lean and obese mice expressed hepcidin at comparable levels; however, isolated macrophages from the stromal vascular fraction expressed higher hepcidin levels. Adipose tissues from obese mice displayed increased tfR2 expression and the presence of iron. Our results indicate that IL-6 and iron may affect the signaling pathways governing hepcidin expression. Thus, the mice fed HFD for 24 wk represent a suitable model for the study of obesity-linked hepcidin alterations. In addition, hepcidin may play local roles in controlling iron availability and interfering with inflammation in adipose tissue.

  4. CETP Expression Protects Female Mice from Obesity-Induced Decline in Exercise Capacity.

    David A Cappel

    Full Text Available Pharmacological approaches to reduce obesity have not resulted in dramatic reductions in the risk of coronary heart disease (CHD. Exercise, in contrast, reduces CHD risk even in the setting of obesity. Cholesteryl Ester Transfer Protein (CETP is a lipid transfer protein that shuttles lipids between serum lipoproteins and tissues. There are sexual-dimorphisms in the effects of CETP in humans. Mice naturally lack CETP, but we previously reported that transgenic expression of CETP increases muscle glycolysis in fasting and protects against insulin resistance with high-fat diet (HFD feeding in female but not male mice. Since glycolysis provides an important energy source for working muscle, we aimed to define if CETP expression protects against the decline in exercise capacity associated with obesity. We measured exercise capacity in female mice that were fed a chow diet and then switched to a HFD. There was no difference in exercise capacity between lean, chow-fed CETP female mice and their non-transgenic littermates. Female CETP transgenic mice were relatively protected against the decline in exercise capacity caused by obesity compared to WT. Despite gaining similar fat mass after 6 weeks of HFD-feeding, female CETP mice showed a nearly two-fold increase in run distance compared to WT. After an additional 6 weeks of HFD-feeding, mice were subjected to a final exercise bout and muscle mitochondria were isolated. We found that improved exercise capacity in CETP mice corresponded with increased muscle mitochondrial oxidative capacity, and increased expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α. These results suggest that CETP can protect against the obesity-induced impairment in exercise capacity and may be a target to improve exercise capacity in the context of obesity.

  5. CETP Expression Protects Female Mice from Obesity-Induced Decline in Exercise Capacity.

    Cappel, David A; Lantier, Louise; Palmisano, Brian T; Wasserman, David H; Stafford, John M


    Pharmacological approaches to reduce obesity have not resulted in dramatic reductions in the risk of coronary heart disease (CHD). Exercise, in contrast, reduces CHD risk even in the setting of obesity. Cholesteryl Ester Transfer Protein (CETP) is a lipid transfer protein that shuttles lipids between serum lipoproteins and tissues. There are sexual-dimorphisms in the effects of CETP in humans. Mice naturally lack CETP, but we previously reported that transgenic expression of CETP increases muscle glycolysis in fasting and protects against insulin resistance with high-fat diet (HFD) feeding in female but not male mice. Since glycolysis provides an important energy source for working muscle, we aimed to define if CETP expression protects against the decline in exercise capacity associated with obesity. We measured exercise capacity in female mice that were fed a chow diet and then switched to a HFD. There was no difference in exercise capacity between lean, chow-fed CETP female mice and their non-transgenic littermates. Female CETP transgenic mice were relatively protected against the decline in exercise capacity caused by obesity compared to WT. Despite gaining similar fat mass after 6 weeks of HFD-feeding, female CETP mice showed a nearly two-fold increase in run distance compared to WT. After an additional 6 weeks of HFD-feeding, mice were subjected to a final exercise bout and muscle mitochondria were isolated. We found that improved exercise capacity in CETP mice corresponded with increased muscle mitochondrial oxidative capacity, and increased expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). These results suggest that CETP can protect against the obesity-induced impairment in exercise capacity and may be a target to improve exercise capacity in the context of obesity.

  6. Spatiotemporal expression of PSD-95 in Fmr1 knockout mice brain.

    Zhu, Zhi-Wei; Xu, Qin; Zhao, Zheng-Yan; Gu, Wei-Zhong; Wu, Ding-Wen


    To investigate and compare the spatial and temporal expression of post-synaptic density-95 (PSD-95) in Fmr1 knockout mice (the animal model of fragile X syndrome, FXS) and wild-type mice brain, on postnatal day 7 (P7), P14, P21, P28 and P90, mice from each group were decapitated, and three principal brain regions (cerebral cortex, hippocampus and cerebellum) were obtained and stored for later experiments. PSD-95 mRNA in the three brain areas was analyzed with quantitative RT-PCR. PSD-95 protein was measured by immunohistochemical staining and Western blot. In the three principal brain areas of Fmr1 knockout mice and wild-type mice, the expression of PSD-95 mRNA and protein were detected at the lowest levels on P7, and then significantly increased on P14, reaching the peak levels in adolescents or adults. Moreover, it was found that PSD-95 mRNA and protein in the hippocampus were significantly decreased in Fmr1 knockout mice during the developmental period (P7, P14, P21 and P28) as well as at adulthood (P90) (P PSD-95 in the cortex and cerebellum between Fmr1 knockout and wild mice. The expression of PSD-95 in the hippocampus might be regulated by fragile X mental retardation protein (FMRP) during mice early developmental and adult periods. It is suggested that impairment of PSD-95 is possibly involved in hippocampal-dependent learning defects, which are common in people with FXS. © 2010 Japanese Society of Neuropathology.

  7. Nuclear Expression of a Mitochondrial DNA Gene: Mitochondrial Targeting of Allotopically Expressed Mutant ATP6 in Transgenic Mice

    David A. Dunn


    Full Text Available Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M or wildtype (A6W mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P0.05. This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

  8. TGF beta 2 mRNA expression and pregnancy failure in mice.

    Gorivodsky, M; Torchinsky, A; Zemliak, I; Savion, S; Fein, A; Toder, V


    We describe here a pattern of transforming growth factor (TGF) beta2 mRNA expression at the fetomaternal interface in mice with high rate of resorptions as well as its expression following maternal immunopotentiation. TGF beta 2 mRNA expression was evaluated in the uteroplacental units of mice with spontaneous (CBA/J x DBA/2J mouse combination) or cyclophosphamide (CP)-induced pregnancy loss. The effect of immunopotentiation on TGF beta 2 mRNA expression was determined in CP-treated females who underwent nonspecific immunostimulation with xenogeneic (rat) leukocytes. A quantitative analysis of TGF beta 2 mRNA level was performed using RNase protection assay. Distribution of TGF beta 2 mRNA transcripts at the fetomaternal interface was studied by in situ hybridization analysis. RNase protection analysis revealed four TGF beta 2 specific mRNA forms (330, 270, 230, and 170 bp) in the uteroplacental units of mice with either normal or decreased reproductive performance. A significant decrease (about 50%) in the level of TGF beta 2 mRNA was registered in the uteroplacental unit of mice with pregnancy loss as compared to the control mice. TGF beta 2 transcripts were abundant in the uterine epithelium and stroma. A specific hybridization signal was detected also in metrial gland cells and it was found to be substantially lower in CP-treated as compared to intact mice. In the resorbing uteroplacental unit, the expression of TGF beta 2 mRNA was completely lost in the uterine epithelium, and the number of TGF beta 2 mRNA-positive metrial gland cells was lower as compared to the control. Immunopotentiation decreased the resorption rate in mice with CP-induced pregnancy loss and caused a dramatic increase in TGF beta 2 mRNA expression: the level of TGF beta 2 mRNA was found to be higher by 2.0-3.2 fold in the uteroplacental unit of immunized as compared to nonimmunized CP-treated mice. These data suggest that distortion of TGF beta 2 expression at the fetomaternal interface

  9. Localization of cystathionine β synthase in mice ovaries and its expression profile during follicular development

    LIANG Rong; YU Wei-dong; DU Jun-bao; YANG Li-jun; SHANG Mei; GUO Jing-zhu


    Background In vitro fertilization (IVF) researches have suggested that cystathionine β synthase (CBS) is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development.Methods We used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 (C57BL × Balb/c) mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development.Results CBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly.Conclusions CBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells.

  10. Thiocyanate supplementation decreases atherosclerotic plaque in mice expressing human myeloperoxidase

    Morgan, P E; Laura, R P; Maki, R A


    Elevated levels of the heme enzyme myeloperoxidase (MPO) are associated with adverse cardiovascular outcomes. MPO predominantly catalyzes formation of the oxidants hypochlorous acid (HOCl) from Cl(-), and hypothiocyanous acid (HOSCN) from SCN(-), with these anions acting as competitive substrates....... HOSCN is a less powerful and more specific oxidant than HOCl, and selectively targets thiols; such damage is largely reversible, unlike much HOCl-induced damage. We hypothesized that increased plasma SCN(-), and hence HOSCN formation instead of HOCl, may decrease artery wall damage. This was examined...... using high-fat fed atherosclerosis-prone LDLR(-/-) mice transgenic for human MPO, with and without SCN(-) (10 mM) added to drinking water. Serum samples, collected fortnightly, were analyzed for cholesterol, triglycerides, thiols, MPO, and SCN(-); study-long exposure was calculated by area under...

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

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


    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.

  12. Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice.

    Yan, Jingbin; Gong, Xiuli; Pan, Shubiao; Guo, Xinbing; Ren, Zhaorui; Zeng, Yitao


    Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 μg/ml in milk.

  13. Effect of Bushenantai Recipe on the Expression of Endometrial LIF in Mice with Embryonic Implantation Dysfunction

    Mingmin ZHANG; Yuqin HUANG; Guijin ZHU; Guangying HUANG; Liping DONG; Jinjin ZHANG


    In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kun- ruing mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of L1F mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve em- bryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.

  14. Nocturnin expression is induced by fasting in the white adipose tissue of restricted fed mice.

    Misty R Gilbert

    Full Text Available The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues. Nocturnin, a circadian deadenylase, is expressed rhythmically in a wide variety of tissues, but we report here that Nocturnin expression is arrhythmic in epididymal white adipose tissue (eWAT of mice housed in 12:12 LD with ad libitum access to food. However, Nocturnin expression becomes rhythmic in eWAT of mice placed on restricted feeding. We show here that Nocturnin's rhythmic expression pattern is not dependent upon feeding, nor is it acutely induced by feeding in the liver or eWAT of ad libitum fed mice. However, Nocturnin is acutely induced by the absence of the expected meal in eWAT of restricted fed mice. A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis. Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes.

  15. Altered Expression of EPO Might Underlie Hepatic Hemangiomas in LRRK2 Knockout Mice.

    Wu, Ben; Xiao, Kaifu; Zhang, Zhuohua; Ma, Long


    Parkinson's disease (PD) is a severe neurodegenerative disorder caused by progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. The molecular mechanism of PD pathogenesis is unclear. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common genetic cause of familial and sporadic PD. However, studies on LRRK2 mutant mice revealed no visible dopaminergic neuronal loss in the midbrain. While surveying a LRRK2 knockout mouse strain, we found that old animals developed age-dependent hepatic vascular growths similar to cavernous hemangiomas. In livers of these hemangioma-positive LRRK2 knockout mice, we detected an increased expression of the HIF-2α protein and significant reactivation of the expression of the HIF-2α target gene erythropoietin (EPO), a finding consistent with a role of the HIF-2α pathway in blood vessel vascularization. We also found that the kidney EPO expression was reduced to 20% of the wild-type level in 18-month-old LRRK2 knockout mice. Unexpectedly, this reduction was restored to wild-type levels when the knockout mice were 22 months to 23 months old, implying a feedback mechanism regulating kidney EPO expression. Our findings reveal a novel function of LRRK2 in regulating EPO expression and imply a potentially novel relationship between PD genes and hematopoiesis.

  16. Effect of Micardis on the Expression of Renal Medulla Aquaporin-2 in Diabetic Mice

    Lijun YAO; Jianqing WANG; Anguo DENG; Jianshe LIU; Hong ZHAO


    In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 (AQP2) was investigated in the renal medullary collecting duct of mice with diabetic nephropathy (DN). Mice were divided into three groups: normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group (2.39±0.11 vs 3.16±0.16, P<0.05). Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system (RAS) is implicated in the pathogenesis of DN by regulating the expression of AQP2.

  17. Increased Skeletal Muscle GLUT4 Expression in Obese Mice After Voluntary Wheel Running Exercise Is Posttranscriptional.

    Gurley, Jami M; Griesel, Beth A; Olson, Ann Louise


    Exercise promotes glucose clearance by increasing skeletal muscle GLUT4-mediated glucose uptake. Importantly, exercise upregulates muscle GLUT4 expression in an insulin-independent manner under conditions of insulin resistance, such as with type 2 diabetes. However, the insulin-independent mechanism responsible for rescued muscle GLUT4 expression is poorly understood. We used voluntary wheel running (VWR) in mice to test the prevailing hypothesis that insulin-independent upregulation of skeletal muscle GLUT4 protein expression with exercise is through increased Glut4 transcription. We demonstrate that 4 weeks of VWR exercise in obese mice rescued high-fat diet-induced decreased muscle GLUT4 protein and improved both fasting plasma insulin and hepatic triacylglyceride levels, but did not rescue muscle Glut4 mRNA. Persistent reduction in Glut4 mRNA suggests that a posttranscriptional mechanism regulated insulin-independent muscle GLUT4 protein expression in response to exercise in lean and obese mice. Reduction of GLUT4 protein in sedentary animals upon treatment with rapamycin revealed mTORC1-dependent GLUT4 regulation. However, no difference in GLUT4 protein expression was observed in VWR-exercised mice treated with either rapamycin or Torin 1, indicating that exercise-dependent regulation on GLUT4 was mTOR independent. The findings provide new insight into the mechanisms responsible for exercise-dependent regulation of GLUT4 in muscle.

  18. Dysregulation of temperature and liver cytokine gene expression in immunodeficient wasted mice

    Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States); Ling-Indeck, L.; Weaver, P. [Loyola Univ. Medical Center, Maywood, IL (United States). Dept. of Pathology; Chang-Liu, Chin-Mei; Strezoska, V.; Heckert, B. [Argonne National Lab., IL (United States). Center for Mechanistic Biology and Biotechnology; Woloschak, G.E. [Loyola Univ. Medical Center, Maywood, IL (United States). Dept. of Pathology]|[Argonne National Lab., IL (United States). Center for Mechanistic Biology and Biotechnology


    Wasted mice bear the spontaneous autosomal recessive mutation wst/wst; this genotype is associated with weight loss beginning at 21 days of age, neurologic dysfunction, immunodeficiency at mucosal sites, and increased sensitivity to the killing effects of ionizing radiation. The pathology underlying the disease symptoms is unknown. Experiments reported here were designed to examine thermoregulation and liver expression of specific cytokines in wasted mice and in littermate and parental controls. Our experiments found that wasted mice begin to show a drop in body temperature at 21-23 days following birth, continuing until death at the age of 28 days. Concomitant with that, livers from wasted mice expressed increased amounts of mRNAs specific for cytokines IL,6 and IL-1, the acute phase reactant C-reactive protein, c-jun, and apoptosis-associated Rp-8 when compared to littermate and parental control animals. Levels of {beta}-transforming growth factor (TGF), c-fos, proliferating cell nuclear antigen (PCNA), and ornithine amino transferase (OAT) transcripts were the same in livers from wasted mice and controls. These results suggest a relationship between an acute phase reactant response in wasted mice and temperature dysregulation.

  19. Collagenlα1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice


    Osteoblasts participate in bone formation,bone mineralization,osteoclast differentiation and many pathological processes.To study the function of genes in osteoblasts using Cre-LoxP system,we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlal (Coilal) promoter(Coilatl-Cre).Two founders were identified by genomic PCR from 16 offsprings.and the integration efficiency is 12.5%.In order tO determine the tissue distribution and the activity of Cre rccombinase in the transgenic mice,the Collal-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4co/co).Multiple tissue PCR of Collal-Cre;Smad4co/+mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon.LacZ staining in the Coilal-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5.Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice.All these data indicated that the Collal-Cre transgenic mice could Serve as a valuabletool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.

  20. GFAP expression and social deficits in transgenic mice overexpressing human sAPPα

    Bailey, Antoinette R; Hou, Huayan; Song, Min; Obregon, Demian F; Portis, Samantha; Barger, Steven; Shytle, Doug; Stock, Saundra; Mori, Takashi; Sanberg, Paul G; Murphy, Tanya; Tan, Jun


    Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed β-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment. PMID:23840007

  1. Expression of JunB Induced by X-rays in Mice



    To explore JunB gene expression in spleen cells of mice after the whole body irradiation as well as in normal hematopoietic and leukemia cells in the primary culture after different dosages of X-ray irradiation. Methods Spleen cells were isolated from the mice irradiated with 3 Gy X-rays. Primary cultured cells from mice were incubated in different intervals after X-irradiation at different dosages. Total RNA was extracted from the cells and the fluctuation of JunB mRNA level was assessed by the RNA ratio of JunB/β-actin measured by quantitative Northern blot hybridization. Results After the mice were exposed to 3 Gy X-rays irradiation, JunB expression in spleen cells was remarkably and rapidly increased, and reached its peak 0.5 h later in C3H/He mice and 1 h later in Balb/c mice. In the primary culture of normal spleen and leukemia cells, JunB mRNA levels increased 30 min after irradiation. The enhanced levels of JunB mRNA were returned to a normal level within 240 min after irradiation. Conclusions JunB gene is responsive to ionizing irradiation and is induced at immediate-early phase after the stimulation. This suggests that the JunB gene plays an important role in the early process of the cells against radiation.

  2. Constriction velocities of renal afferent and efferent arterioles of mice are not related to SMB expression.

    Patzak, Andreas; Petzhold, Daria; Wronski, Thomas; Martinka, Peter; Babu, Gopal J; Periasamy, Muthu; Haase, Hannelore; Morano, Ingo


    Constriction of renal arterioles contributes significantly to the control of perfusion and glomerular filtration. Afferent but not efferent arterioles express smooth muscle myosin heavy chain B (SMB) (with a 5'-insert of seven amino acids). The aim of the present study was to investigate (1) the constriction characteristics of afferent and efferent arterioles under physiologic load and (2) whether expression of SMB may causally contribute to these constriction characteristics. We compared constriction parameters [constriction amplitude, maximal rate of constriction velocity ("dc/dt(max)"), and time to half-maximal constriction (t(1/2)) of in vitro perfused renal afferent and efferent arterioles of wild-type (smb(+/+)] and homozygous SMB knockout [smb(-/-)] mice upon stimulation with angiotensin II (Ang II) (10(-8) mol/L) and potassium chloride (KCl) (100 mmol/L). SMB expression was investigated by double-labeling immunofluorescence. Contraction amplitude and dc/dt(max) of mouse afferent arterioles upon Ang II stimulation were significantly greater compared to efferent arterioles. However, constriction amplitudes, dc/dt(max), and t(1/2) of afferent as well as efferent arterioles upon Ang II stimulation were similar in smb(+/+) and smb(-/-) mice. Constriction amplitudes upon KCl stimulation of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. Furthermore, KCl-induced dc/dt(max) and t(1/2) of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. SMB expression could be detected in afferent but not efferent arterioles in smb(+/+) mice. No SMB expression in either arteriole could be observed in smb(-/-) mice. Our results suggest that the presence of different alternatively 5'-spliced smooth muscle-myosin heavy chain (SM-MHC) isoforms does not dominate the different contractile features of physiologically loaded renal afferent or efferent arterioles.

  3. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice.

    Dejia Li

    Full Text Available Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

  4. Lactate Transporters Expression in Tumor of Balb/c Mice Bearing Breast Cancer after Endurance Training

    M Aveseh


    Full Text Available Background & aim: Changes in the metabolism of cancer cells plays a major role in the survival and their expansion. The aim of this study was to determine expression of lactate transmitters in Balb/c mice with breast cancer after endurance training. Methods: In this experimental study twenty-five Balb C mice were randomly divided into two groups of breast cancer control (N=13 and breast cancer training (N=12. Breast cancer was induced in mammary fat pad by injection of cancer cells (MC4L2 in mice and endurance training protocol was applied for 7 weeks in the experimental group. Tumor volume and MCT1, MCT4, and CD147 expression were measured by micro digital caliper and western blotting technique respectively. Data were analyzed statistically using Student t and Pearson. Results: Significant decreases was found in weight and CD147 expression of tumor after 7 weeks of endurance training in the exercise group compared to the control group. No significant differences were seen in MCT4 expression and tumor volume between the groups (05 / 0p>0.05. Significant correlation was found between tumor MCT1 and CD147 expression (P < 0.05, while the relationship between MCT4 and CD147 expression in tumors was not statistically significant. Conclusion: Endurance training can reduce lactate metabolism in cancer cells through suppression of lactate transporters expression and provides a useful tool in breast cancer treatment or prevention.

  5. Expression of recombinant human lysozyme in the milk of transgenic mice

    YU Zhengquan; FAN Baoliang; DAI Yunping; ZHENG Ming; NIU Huiling; WANG Meili; WANG Lili; FEI Jing; LI Ning


    Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal ofimproving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic micewas 0.2 mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.

  6. bcl-xl over-expression in transgenic mice reduces cerebral ischemia/reperfusion injury

    Furong Wang; Yongsheng Jiang; Yan Liu; Wenwu Xiao; Suming Zhang


    BACKGROUND: Basal cell lymphoma-extra large (bcl-xl) can inhibit neuronal apoptosis by stabilizing the mitochondrial membrane and suppressing cytochrome C release into the cytoplasm. OBJECTIVE: This study aimed to further investigate the cascade reaction pathway of cellular apoptosis. We established an ischemia/dreperfusion model by middle cerebral artery occlusion (MCAO) in transgenic and wild-type mice, and observed changes in the number and distribution of apoptotic neural cells, differences in cerebral infarct volume, in neurological function score, and in cytochrome C expression in the ischemic cerebral cortex, at different time points, DESIGN AND SETTING: The present gene engineering and cell biology experiment was performed at the Laboratory of Biology, Hubei Academy of Agricultural Sciences and at the Laboratory of Immunology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Male bcl-xl over-expression Kunming mice aged 8 weeks and age-matched male wild-type mice were used for this study. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) kits were purchased from Boliman, France. Cytochrome C antibody and Bcl-x immunohistochemical kit were purchased from PharMingen, USA and Santa Cruz Biotechnology, USA, respectively. METHODS: Following MCAO and reperfusion, apoptosis in the ischemic cerebral cortex was detected by the TUNEL assay. Prior to MCAO and 3 hours after reperfusion, the Bcl-xl protein level in the ischemic cerebral cortex was measured by immunohistochemistry. At 3, 6, 12 and 24 hours after reperfusion, the level of cytochrome C in the ischemic cerebral cortex was examined by western blot analysis. Subsequent to MCAO, cerebral infarct volume measurement and neurological examination were performed. MAIN OUTCOME MEASURES: Neural cell apoptosis and cytochrome C expression in the ischemic cerebral cortex; cerebral infarct volume and neurological function score. RESULTS: Twenty-four hours after

  7. Insulin expression in livers of diabetic mice mediated by hydrodynamics-based administration

    Chen-Xia He; Da-Ru Lu; Jing-Lun Xue; Ding Shi; Wen-Jun Wu; You-Fa Ding; Deng-Min Feng; Bin Lu; Hao-Ming Chen; Ji-Hua Yao; Qi Shen


    AIM: Transfer and expression of insulin gene in vivo are an alternative strategy to improve glycemic control in type 1 diabetes. Hydrodynamics-based procedure has been proved to be very efficient to transfer naked DNA to mouse livers. The basal hepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resumption of glycemic control in type 1diabetic mice.METHODS: Engineered insulin cDNA was inserted into plasmid vectors under a ClMlV promoter, and transferred into STZ induced diabetic mice by hydrodynamic procedure.Glucose levels, body weight of treated mice, insulin levels,immunohistology of the liver, and quantity of insulin mRNA in the liver were assayed to identify the improvement of hyperglycemic complication after plasmid administration.Sleeping Beauty, a transposon system, was also used to prolong the insulin expression in the liver.RESULTS: After plasmid administration, Plasma insulin was significantly increased in the diabetic mice and the livers were insulin-positive by immunostaining. At the same time the hyperglycemic complication was improved. The blood glucose levels of mice were reduced to normal. Glucose tolerance of the treated diabetic mice was improved. Body weight loss was also ameliorated. The rapid tail vein injection did not cause any fatal result.CONCLUSION: Our results suggested that insulin gene could be efficiently transferred into the livers of diabetic mice via rapid tail vein injection and it resulted in high level of insulin expression. The basal hepatic insulin production mediated by hydrodynamics-based administration improved the glycemic control in type 1 diabetes dramatically and ameliorated diabetic syndromes. Hydrodynamics-based administration offers a simple and efficient way in the study of gene therapy for type 1 diabetes.

  8. Immunoglobulin gene expression and regulation of rearrangement in kappa transgenic mice

    Ritchie, K.A.


    Transgenic mice were produced by microinjection of the functionally rearranged immunoglobulin kappa gene from the myeloma MOPC-21 into the male pronucleus of fertilized mouse eggs, and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were detected by hybridizing tail DNA dots with radioactively labelled pBR322 plasmid DNA, which detects pBR322 sequences left as a tag on the microinjected DNA. Mice that integrated the injected gene (six males) were mated and the DNA, RNA and serum kappa chains of their offspring were analyzed. A rabbit anti-mouse kappa chain antiserum was also produced for use in detection of mouse kappa chains on protein blots. Hybridomas were produced from the spleen cells of these kappa transgenic mice to immortalize representative B cells and to investigate expression of the transgenic kappa gene, its effect on allelic exclusion, and its effect on the control of light chain gene rearrangement and expression. The results show that the microinjected DNA is integrated as concatamers in unique single or, rarely, two separate sites in the genome. The concatamers are composed of several copies (16 to 64) of injected DNA arranged in a head to tail fashion. The transgene is expressed into protein normally and in a tissue specific fashion. For the first time in these transgenic mice, all tissues contain a functionally rearranged and potentially expressible immunoglobulin gene. The transgene is expressed only in B cells and not in hepatocytes, for example. This indicates that rearrangement of immunoglobulin genes is necessary but not sufficient for the tissue specific expression of these genes by B cells.

  9. Function and expression of cystic fibrosis transmembrane conductance regulator after small intestinal transplantation in mice.

    Penghong Song

    Full Text Available The secretion function of intestinal graft is one of the most important factors for successful intestinal transplantation. Cystic fibrosis transmembrane conductance regulator (CFTR mediates HCO3(- and Cl(- secretions in intestinal epithelial cells. In this study, we made investigation on the expression and function of CFTR in an experimental model of murine small intestinal transplantation. Heterotopic intestinal transplantations were performed in syngeneic mice. The mRNA and protein expressions of CFTR were analyzed by real time PCR and western blot. Murine intestinal mucosal HCO3(- and Cl(- secretions were examined in vitro in Ussing chambers by the pH stat and short circuit current (I(sc techniques. The results showed that forskolin, an activator of CFTR, stimulated jejunal mucosal epithelial HCO3(- and Cl(- secretions in mice, but forskolin-stimulated HCO3(- and Cl(- secretions in donor and recipient jejunal mucosae of mice after heterotopic jejunal transplantation were markedly decreased, compared with controls (P<0.001. The mRNA and protein expression levels of CFTR in donor and recipient jejunal mucosae of mice were also markedly lower than those in controls (P<0.001, and the mRNA and protein expression levels of tumor necrosis factor α (TNFα were markedly increased in donor jejunal mucosae of mice (P<0.001, compared with controls. Further experiments showed that TNFα down-regulated the expression of CFTR mRNA in murine jejunal mucosa. In conclusion, after intestinal transplantation, the function of CFTR was impaired, and its mRNA and protein expressions were down-regulated, which may be induced by TNFα.

  10. Electroacupuncture reduces Aβ production and BACE1 expression in SAMP8 mice

    Weiguo eDong


    Full Text Available Electroacupuncture (EA has been reported to have beneficial effects on Alzheimer's disease (AD. BACE1 (β-site amyloid precursor protein-cleaving enzyme 1 is involved in the abnormal production of amyloid-β plaque (Aβ, a hallmark of AD pathophysiology. Thus, the present study investigated the effects of EA on memory impairment, Aβ production, and BACE1 expression in SAMP8 mice. We found that EA improved spatial learning and memory impairment of SAMP8 mice. Furthermore, EA attenuated Aβ production and repressed the expression of BACE1 in the hippocampus of SAMP8 mice. Taken together, our results suggest that EA could have a potential therapeutic application in AD and that BACE1 may be an important target of EA in the treatment of AD.

  11. Expression of truncated PITX3 in the developing lens leads to microphthalmia and aphakia in mice.

    Kenta Wada

    Full Text Available Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3 as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of

  12. Conditional Expression of Wnt4 during Chondrogenesis Leads to Dwarfism in Mice

    Lee, Hu-Hui; Behringer, Richard R.


    Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26floxneoWnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype. PMID:17505543

  13. Expression of truncated PITX3 in the developing lens leads to microphthalmia and aphakia in mice.

    Wada, Kenta; Matsushima, Yoshibumi; Tada, Tomoki; Hasegawa, Sayaka; Obara, Yo; Yoshizawa, Yasuhiro; Takahashi, Gou; Hiai, Hiroshi; Shimanuki, Midori; Suzuki, Sari; Saitou, Junichi; Yamamoto, Naoki; Ichikawa, Masumi; Watanabe, Kei; Kikkawa, Yoshiaki


    Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3) as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain) as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of downstream targets and

  14. Conditional expression of Wnt4 during chondrogenesis leads to dwarfism in mice.

    Hu-Hui Lee

    Full Text Available Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26 locus by gene targeting in embryonic stem (ES cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26(floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26(floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26(floxneoWnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF. These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype.

  15. Efficacious cellular codelivery of doxorubicin and EGFP siRNA mediated by the composition of PLGA and PEI protected gold nanoparticles.

    Kumar, Krishan; Vulugundam, Gururaja; Jaiswal, Pradeep Kumar; Shyamlal, Bharti Rajesh Kumar; Chaudhary, Sandeep


    This study reports the simultaneous delivery of EGFP siRNA and the chemotherapeutic drug, doxorubicin by means of the composition that results from the electrostatic interaction between positively charged siRNA-complexes of gold nanoparticles (AuNPs) capped with PEI, 25kDa (P25-AuNPs) and negatively charged carboxymethyl cellulose formulated PLGA nanoparticles loaded with doxorubicin. The nanoparticles and their facile interaction were studied by means of dynamic light scattering (DLS), zeta potential, transmission electron microscopic (TEM) measurements. The flow cytometric and confocal microscopic analysis evidenced the simultaneous internalization of both labelled siRNA and doxorubin into around 55% of the HeLa cancer cell population. Fluorescence microscopic studies enabled the visual analysis of EGFP expressing HeLa cells which suggested that the composition mediated codelivery resulted in a substantial downregulation of EGFP expression and intracellular accumulation of doxorubicin. Interestingly, codelivery treatment resulted in an increased cellular delivery of doxorubicin when compared to PLGA-DOX alone treatment. On the other hand, the activity of siRNA complexes of PEI-AuNPs was completely retained even when they were part of composition. The results suggest that this formulation can serve as promising tool for delivery applications in combinatorial anticancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice.

    Zang, Zhi-Jun; Fang, You-Qiang; Ji, Su-Yun; Gao, Yong; Zhu, Yuan-Qiang; Xia, Ting-Ting; Jiang, Mei-Hua; Zhang, Ya-Nan


    Formaldehyde, a ubiquitous environmental pollutant, is used extensively and has been proved to impair male reproduction in mammals. However, no trials have explored whether formaldehyde affects sexual function. To evaluate the effect of long-term formaldehyde exposure on sexual behavior and to investigate the potential mechanism. Forty C57BL/6 male mice were randomly allocated to four equally sized groups. Mice were exposed to formaldehyde at a dose of 0 (control), 0.5, 5.0, or 10.0 mg/m(3) by inhalation for 60 days. Sexual behavior, body and reproductive organ weights, testosterone concentration in serum and testicular tissue, expression of steroidogenic enzymes, quality of sperm, and testicular structure were measured. Formaldehyde inhibited sexual behavior and decreased reproductive organ weights in mice. Serum testosterone levels and intratesticular testosterone concentrations were decreased in the formaldehyde-treated groups. Expression levels of steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase (3β-HSD), also were decreased in the testes of mice exposed to formaldehyde. Moreover, the structure of seminiferous tubules was destroyed and sperm quality decreased after formaldehyde exposure. In addition, the results indicated that the effects of formaldehyde were dose dependent. Efforts should be undertaken to decrease impairment of sexual function caused by formaldehyde exposure. The relatively small sample might have affected the outcomes. Further experiments are needed to study the mechanism of action of formaldehyde. Exposure to formaldehyde gas inhibited sexual behavior, caused reproductive organ atrophy, and impaired spermatogenesis in male mice, which might have been induced by suppressed expression of steroidogenic enzymes in Leydig cells and decreased testosterone synthesis. Zang Z-J, Fang Y-Q, Ji S-Y, et al. Formaldehyde Inhibits Sexual


    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  18. Postnatal events in intestinal gene expression and splenic cell composition is altered in NOD mice

    Damlund, Dina Silke Malling; Metzdorff, Stine Broeng; Kristensen, Matilde Bylov


    , cellular composition in spleen and liver. At PND1 and 2, the number of Ly-6G and CD11b positive cells in NOD mice was significantly (p=0.05) higher as compared to C57/bl6. Furthermore, gene expression analyses of liver, spleen and intestine showed differences between the two mouse strains in the early...

  19. Montelukast modulates lung CysLT1 receptor expression and eosinophilic inflammation in asthmatic mice

    Yan-jun ZHANG; Lei ZHANG; Shao-bin WANG; Hua-hao SHEN; Er-qing WEI


    AIM: To determine the expressions of cysteinyl leukotriene receptors, CysLT1 and CysLT2, in airway eosinophilic inflammation of OVA-induced asthmatic mice and the modulation by montelukast, a CysLT1 receptor antagonist.METHODS: Asthma model was induced by chronic exposure to ovalbumin (OVA) in C57BL/6 mice. The eosinophils in bronchoalveolar lavage (BAL) fluid and lung tissues were counted, IL-5 level in BAL fluid was measured,and CysLT1 and CysLT2 receptor mRNA expressions were detected by semi-quantitative RT-PCR. RESULTS:Montelukast (6 mg/kg, once per day for 20 d) significantly suppressed the increased eosinophils in BAL fluid and lung tissue, and increased IL-5 level in BAL fluid in OVA challenged mice. OVA challenge increased CysLT1 but decreased CysLT2 receptor mRNA expression. Montelukast inhibited the increased CysLT1 but not the reduced CysLT2 expression after OVA challenge. CONCLUSION: CysLT receptors are modulated immunologically, and montelukast inhibits up-regulation of CysLT1 receptor and airway eosinophilic inflammation in asthmatic mice.

  20. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice

    Janssen, KP; El Marjou, F; Pinto, D; Sastre, X; Rouillard, D; Fouquet, C; Soussi, T; Louvard, D; Robine, S


    Background & Aims: Ras oncoproteins are mutated in about 50% of human colorectal cancers, but their precise role in tumor initiation or progression is still unclear. Methods: This study presents transgenic mice that express K-ras(V12G), the most frequent oncogenic mutation in human tumors, under con


    The effects of hyperthermia on spermatogenesis, apoptosis, gene expression and fertility in adult male miceJohn C. Rockett1, Faye L. Mapp1, J. Brian Garges1, J. Christopher Luft1, Chisato Mori2 and David J. Dix1.1Reproductive Toxicology Division, National Health and Envir...

  2. Effect of Micardis on the expression of renal medulla aquaporin-2 in diabetic mice.

    Yao, Lijun; Wang, Jianqing; Deng, Anguo; Liu, Jianshe; Zhao, Hong


    In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 (AQP2) was investigated in the renal medullary collecting duct of mice with diabetic nephropathy (DN). Mice were divided into three groups: normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group (2.39+/-0.11 vs 3.16+/-0.16, PMicardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system (RAS) is implicated in the pathogenesis of DN by regulating the expression of AQP2.

  3. Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice

    Liu Zhihui


    Full Text Available Abstract Background Interleukin 1 beta (IL-1β plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1β in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1β gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. Results In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1β mRNA and pro-IL-1β protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. Conclusion Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo.

  4. Gene expression alterations in brains of mice infected with three strains of scrapie

    Race Richard E


    Full Text Available Abstract Background Transmissible spongiform encephalopathies (TSEs or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD in humans, bovine spongiform encephalopathy (BSE in cattle, chronic wasting disease (CWD in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML. Results In symptomatic mice, over 400 significant gene expression alterations were identified. In contrast, only 22 genes showed significant alteration in the pre-symptomatic animals. We also identified genes that showed significant differences in alterations in gene expression between strains. Genes identified in this study encode proteins that are involved in many cellular processes including protein folding, endosome/lysosome function, immunity, synapse function, metal ion binding, calcium regulation and cytoskeletal function. Conclusion These studies shed light on the complex molecular events that occur during prion disease, and identify genes whose further study may yield new insights into strain specific neuropathogenesis and ante-mortem tests for TSEs.

  5. Volatile of alkyd varnish inhibits the expression of neuronal growth associated protein-43 in mice

    Qian Huang; Hongxia Wang; Wei Zou


    BACKGROUND: Studies have demonstrated that coating materials used commonly in the interior decoration contain volatile of alkyd varnish, which has obvious effects on the structure of endothelial cells of respiratory tract.OBJECTIVE: To observe the effects of volatile of alkyd varnish in the decoration materials on the expression of neuronal growth associated protein-43 (GAP-43) in mice, and to analyze the influencing mechanism of interior environmental pollutants for brain mechanism.DESIGN: A randomized grouping and controlled experiment.SETTING: College of Life Science, Liaoning Normal University.MATERIALS: This study was carried out in the College of Life Science, Liaoning Normal University between February and December 2006. Twenty one-month-old Kunming mice, weighing (20 + 2) g, male and female in half, were involved in this study. The involved mice were divided into 2 groups by random lot method: chronic poisoning group (n =10) and control group (n =10). Alkyd varnish used commonly for house decoration was purchased from Furnishing World of Liberation Plaza of Dalian City. Alkyd varnish used commonly for house decoration was purchased from Furnishing World of Liberation Plaza of Dalian City. Rabbit GAP-43 polyclonal antibody, rat P -actin monoclonal antibody, goat anti-rat IgG-HRP and goat ant-rabbit IgG-HRP were purchased from Boster Company (Wuhan). ECL was purchased from Amersham Company (Britain), other related reagents were all purchased from Sigma Company and Promega Company(USA). All the other reagents were home-made analytical pure.METHODS:①Poisoning test: The mice were poisoned by static inhalation poisoning method. Mice in the chronic poisoning group were placed in the 0.024 m3 poisoning cabinet. Alkyd varnish (8g,3-time dosage of house decoration) was daily spread once on a 40 cm×21 cm kraft paper evenly. Mice were poisoned for 14 hours within 3 weeks successively. Mice in the control group were placed in the same environment without alkyd

  6. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    Fromont-Racine, M; Bucchini, D; Madsen, O;


    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific tra...... of the transgene was observed in cell types other than beta-islet cells.......Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...

  7. Increased expression of tyrosine hydroxylase and anomalous neurites in catecholaminergic neurons of ATF-2 null mice.

    Kojima, Masayo; Suzuki, Takahiro; Maekawa, Toshio; Ishii, Shunsuke; Sumi-Ichinose, Chiho; Nomura, Takahide; Ichinose, Hiroshi


    ATF-2/CRE-BP1 was originally identified as a cAMP-responsive element (CRE) binding protein abundant in the brain. We previously reported that phosphorylation of ATF-2 increased the expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme for catecholamine biosynthesis, directly acting on the CRE in the promoter region of the TH gene in PC12D cells (Suzuki et al. [2002] J. Biol. Chem. 277:40768-40774). To examine the role of ATF-2 on transcriptional control of the TH gene in the brain, we investigated the TH expression in ATF-2-/- mice. We found that TH expression was greatly increased in medulla oblongata and locus ceruleus of the ATF-2-deficient embryos. Ectopic expression of TH was observed in the raphe magnus nucleus, where serotonergic neural cell bodies are located. Interestingly, A10 dorsal neurons were lost in the embryos of ATF-2-/- mice. There was no difference in the TH immunoreactivity in the olfactory bulb. The data showed that alteration in TH expression by absence of ATF-2 gradually declined from caudal to rostral part of the brain. We also found anomalous neurite extension in catecholaminergic neurons of ATF-2 null mice, i.e., increased dendritic arborization and shortened axons. These data suggest that ATF-2 plays critical roles for proper expression of the TH gene and for neurite extension of catecholaminergic neurons, possibly through a repressor-like action. (c) 2007 Wiley-Liss, Inc.

  8. Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice.

    Lafond, R E; Giammalvo, J T; Norkin, L C


    The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses the Int-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.

  9. Transgenic mice expressing mutant Pinin exhibit muscular dystrophy, nebulin deficiency and elevated expression of slow-type muscle fiber genes

    Wu, Hsu-Pin; Hsu, Shu-Yuan [Department of Anatomy, Chang Gung University Medical College, Taiwan (China); Wu, Wen-Ai; Hu, Ji-Wei [Transgenic Mouse Core Laboratory, Chang Gung University, Taiwan (China); Ouyang, Pin, E-mail: [Department of Anatomy, Chang Gung University Medical College, Taiwan (China); Transgenic Mouse Core Laboratory, Chang Gung University, Taiwan (China); Molecular Medicine Research Center, Chang Gung University, Taiwan (China)


    Highlights: •Pnn CCD domain functions as a dominant negative mutant regulating Pnn expression and function. •Pnn CCD mutant Tg mice have a muscle wasting phenotype during development and show dystrophic histological features. •Pnn mutant muscles are susceptible to slow fiber type gene transition and NEB reduction. •The Tg mouse generated by overexpression of the Pnn CCD domain displays many characteristics resembling NEB{sup +/−} mice. -- Abstract: Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species. The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity.

  10. Improved Retroviral Vector Design Results in Sustained Expression after Adult Gene Therapy in Mucopolysaccharidosis I Mice

    Herati, Ramin Sedaghat; Ma, Xiucui; Tittiger, Mindy; Ohlemiller, Kevin K; Kovacs, Attila; Ponder, Katherine P.


    Background Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to α-L-iduronidase (IDUA) deficiency that results in the accumulation of glycosaminoglycans (GAG). Gene therapy can reduce most clinical manifestations, but mice that receive transfer as adults lose expression unless they receive immunosuppression. Increasing liver specificity of transgene expression has reduced immune responses to other genes. Methods A gamma retroviral vector was generated with a liver-specific human α1-antitrypsin promoter and the canine IDUA cDNA inverted relative to the retroviral long-terminal repeat. Adult MPS I mice received the vector intravenously at 6 weeks of age and were assessed for expression via serial serum IDUA assays. Functional testing and organ analysis were performed at 8 months. Results This vector resulted in high specificity of expression in liver, and serum IDUA activity was stable in 90% of animals. Although the average serum IDUA activity was relatively low at 12.6 ± 8.1 units/mL in mice with stable expression, a relatively high percentage of enzyme contained the mannose 6-phosphorylation necessary for uptake by other cells. At 6.5 months after transduction, most organs had high IDUA activity and normalized GAG levels. There was complete correction of hearing and vision abnormalities and significant improvements in bone, although the aorta was refractory to treatment. Conclusions Stable expression of IDUA in adult MPS I mice can be achieved without immunosuppression by modifying the vector to reduce expression in the spleen. This approach may be effective in patients with MPS I or other lysosomal storage diseases. PMID:18613275

  11. Expression of anion exchanger 3 influences respiratory rate in awake and isoflurane anesthetized mice.

    Meier, S; Hübner, C A; Groeben, H; Peters, J; Bingmann, D; Wiemann, M


    The anion exchanger 3 (AE3) is involved in neuronal pH regulation of which may include chemosensitive neurons. Here we examined the effect of AE3 expression on respiratory rate (RR) in vivo. AE3 knockout (KO, n=5) and wild type (WT, n=6) mice were subjected to body plethysmography, both while awake and during isoflurane anesthesia. RR was significantly lower in awake AE3 KO (162+/-7SE min(-1)) than in WT mice (212+/-20 min(-1), P=0.036). The same was found during isoflurane anesthesia at 0.5 MAC (KO: 123+/-9 min(-1), WT: 168+/-15 min(-1), P=0.026) and 1.0 MAC (KO: 51+/-6 min(-1), WT: 94+/-6 min(-1), P=0.001). Hypercapnia (5% CO2) increased RR in awake and decreased RR in nesthetized (1.0 MAC) mice, whereby relative changes were larger in AE3 KO mice. Recovery from isoflurane anesthesia in respect to RR regaining baseline values was more pronounced in AE3 KO. Results show that AE3 expression profoundly influences control of breathing in mice.

  12. Diversity of TCRs on natural Foxp3+ T cells in mice lacking Aire expression.

    Daniely, Danielle; Kern, Joanna; Cebula, Anna; Ignatowicz, Leszek


    Medullary thymic epithelial cells expressing the Aire gene play a critical role in the induction of tolerance to tissue-specific Ags (TSAs). It was postulated that recognition of Aire-controlled TSAs by immature thymocytes results in the selection of natural CD4+Foxp3+ regulatory T cells (Tregs) and enriches this repertoire in self-reactive receptors, contributing to its vast diversity. In this study, we compared the TCRs on individual Tregs in Aire+ and Aire- mice expressing a miniature TCR repertoire (TCRmini) along with GFP driven by the Foxp3 promoter (Foxp3GFP). The Treg TCR repertoires in Aire+ and Aire- TCRminiFoxp3GFP mice were similar and more diverse than their repertoires on CD4+ Foxp3- thymocytes. Further, TCRs found on potentially self-reactive T cells, with an activated phenotype (CD4+Foxp3-CD62Llow) in Aire- TCRminiFoxp3GFP mice, appear distinct from TCRs found on Tregs in Aire+ TCRminiFoxp3GFP mice. Lastly, we found no evidence that TSAs presented by medullary thymic epithelial cells in Aire+TCRmini mice are often recognized as agonists by Treg-derived TCR hybridomas or CD4+CD25+ thymocytes, containing both natural Tregs and precursors. Thus, positive selection and self-reactivity of the global Treg repertoire are not controlled by Aire-dependent TSAs.

  13. Abnormalities in Monocyte Recruitment and Cytokine Expression in Monocyte Chemoattractant Protein 1–deficient Mice

    Lu, Bao; Rutledge, Barbara J.; Gu, Long; Fiorillo, Joseph; Lukacs, Nicholas W.; Kunkel, Steven L.; North, Robert; Gerard, Craig; Rollins, Barrett J.


    Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1–deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1−/− mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1−/− mice, as was expression of IL-4, IL-5, and interferon γ in splenocytes. In contrast, MCP-1−/− mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses. PMID:9463410

  14. Differential protein expression in heart in UT-B null mice with cardiac conduction defects.

    Yu, Hao; Meng, Yan; Wang, Li-Shun; Jin, Xian; Gao, Li-Fang; Zhou, Lei; Ji, Kun; Li, Yang; Zhao, Li-Juan; Chen, Guo-Qiang; Zhao, Xue-Jian; Yang, Baoxue


    Cardiac conduction defects were found in transgenic mice deficient in urea transporter UT-B. To investigate the molecular mechanisms of the conduction defects caused by UT-B deletion, we studied the protein expression profiles of heart tissue (comprising most conduction system) in wild-type versus UT-B null mice at different ages. By two-dimensional electrophoresis-based comparative analysis, we found that more than dozen proteins were modulated (>two-fold) in the myocardium of UT-B null mice. Out of these modulated proteins, troponin T (TNNT2) presented significant changes in UT-B null mice at early stage prior to the development of P-R interval elongation, while the change of atrial natriuretic peptide (ANP) occurred only at late stage in UT-B null mice that had the AV block. These data indicate that UT-B deletion caused the dynamic expression regulation of TNNT2 and ANP, and these proteins may provide new clues to investigate the molecular events involved in cardiac conduction.

  15. The Forkhead Transcription Factor, FOXP3, Is Required for Normal Pituitary Gonadotropin Expression in Mice1

    Jung, Deborah O.; Jasurda, Jake S.; Egashira, Noboru; Ellsworth, Buffy S.


    ABSTRACT The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3sf/Y). Although the gonadotroph cells appear to be intact in Foxp3sf/Y mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3sf/Y males. Treatment of Foxp3sf/Y males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3sf/Y males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3sf/Y males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by

  16. Oral vaccination of mice against rodent malaria with recombinant expressing MSP-119

    Zhi-Hong Zhang; Pei-Hong Jiang; Ning-Jun Li; Mi Shi; Weida Huang


    AIM: To construct the recombinant Lactococcus lactis as oral delivery vaccination against malaria.METHODS: The C-terminal 19-ku fragments of MSP1(MSP-119) of Plasmodium yoelii265-BY was expressed in L. lactis and the recombinant L. lactis was administered orally to BALB/c and C57BL/6 mice. After seven interval vaccinations within 4 wk, the mice were challenged with P.yoelii 265-BY parasites of erythrocytic stage. The protective efficacy of recombinant L.lactiswas evaluated.RESULTS: The peak parasitemias in average for the experiment groups of BALB/c and C57BL/6 mice were 0.8±0.4% and 20.8±26.5%, respectively, and those of their control groups were 12.0±0.8% and 60.8±9.6%, respectively. None of the BALB/c mice in both experimental group and control group died during the experiment.However, all the C57BL/6 mice in the control group died within 23 d and all the vaccinated mice survived well.CONCLUSION: The results imply the potential of recombinant L.lactis as oral delivery vaccination against malaria.

  17. Effects of soft-diet feeding on BDNF expression in hippocampus of mice.

    Yamamoto, Tetsu; Hirayama, Akihiko; Hosoe, Nobuo; Furube, Masaru; Hirano, Shusuke


    Our previous study showed that mice fed a soft diet after weaning had reduced synaptic connections in the hippocampal formation and impaired spatial learning ability after 3 months of age. We hypothesized that soft-diet feeding during development reduced levels of brain-derived neurotrophic factor (BDNF) protein in the hippocampus, resulting in lower synaptic densities in this region. Male pups of C57BL/6 mice were fed either a solid (hard-diet group) or powdered diet (soft-diet group), starting at weaning. Expression of BDNF protein in the hippocampus and cerebral cortex was evaluated quantitatively with enzyme-linked immunosorbent assay (ELISA) at 1, 3 and 6 months of age. Reduction in BDNF protein levels due to soft diet was detected markedly in the hippocampus of 3- and 6-month-old mice. On the other hand, a soft diet showed no significant effect on BDNF content in the cerebral cortex throughout the ages investigated. Immunohistochemistry of hippocampal formation in 3-month-old mice revealed that intensities of BDNF immunoreactivity in the dentate gyrus granule cell layer and CA1 and CA3 pyramidal cell layers appeared diminished in mice fed the soft diet compared with mice fed the hard diet. These results indicate that insufficient mastication activity during development reduces BDNF protein levels in the hippocampus and influences synaptic plasticity in this region.

  18. Number of X-chromosome genes influences social behavior and vasopressin gene expression in mice.

    Cox, Kimberly H; Quinnies, Kayla M; Eschendroeder, Alex; Didrick, Paula M; Eugster, Erica A; Rissman, Emilie F


    Sex differences in behavior are widespread and often caused by hormonal differences between the sexes. In addition to hormones, the composition and numbers of the sex chromosomes also affect a variety of sex differences. In humans, X-chromosome genes are implicated in neurobehavioral disorders (i.e. fragile-X, autism). To investigate the role of X-chromosome genes in social behavior, we used a mouse model that has atypical sex chromosome configurations resembling Turner (45, XO) and Klinefelter syndromes (47, XXY). We examined a number of behaviors in juvenile mice. Mice with only one copy of most X-chromosome genes, regardless of gonadal sex, were less social in dyadic interaction and social preference tasks. In the elevated plus maze, mice with one X-chromosome spent less time in the distal ends of the open arms as compared to mice with two copies of X-chromosome genes. Using qRTPCR, we noted that amygdala from female mice with one X-chromosome had higher expression levels of vasopressin (Avp) as compared to mice in the other groups. Finally, in plasma from girls with Turner syndrome we detected reduced vasopressin (AVP) concentrations as compared to control patients. These novel findings link sex chromosome genes with social behavior via concentrations of AVP in brain, adding to our understanding of sex differences in neurobehavioral disorders.

  19. 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

    黎皓; 唐七义; 张云; 王树蕙; 郭彩云


    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。

  20. Neonatal maternal deprivation response and developmental changes in gene expression revealed by hypothalamic gene expression profiling in mice.

    Feng Ding

    Full Text Available Neonatal feeding problems are observed in several genetic diseases including Prader-Willi syndrome (PWS. Later in life, individuals with PWS develop hyperphagia and obesity due to lack of appetite control. We hypothesized that failure to thrive in infancy and later-onset hyperphagia are related and could be due to a defect in the hypothalamus. In this study, we performed gene expression microarray analysis of the hypothalamic response to maternal deprivation in neonatal wild-type and Snord116del mice, a mouse model for PWS in which a cluster of imprinted C/D box snoRNAs is deleted. The neonatal starvation response in both strains was dramatically different from that reported in adult rodents. Genes that are affected by adult starvation showed no expression change in the hypothalamus of 5 day-old pups after 6 hours of maternal deprivation. Unlike in adult rodents, expression levels of Nanos2 and Pdk4 were increased, and those of Pgpep1, Ndp, Brms1l, Mett10d, and Snx1 were decreased after neonatal deprivation. In addition, we compared hypothalamic gene expression profiles at postnatal days 5 and 13 and observed significant developmental changes. Notably, the gene expression profiles of Snord116del deletion mice and wild-type littermates were very similar at all time points and conditions, arguing against a role of Snord116 in feeding regulation in the neonatal period.

  1. Widespread over-expression of the X chromosome in sterile F₁hybrid mice.

    Jeffrey M Good


    Full Text Available The X chromosome often plays a central role in hybrid male sterility between species, but it is unclear if this reflects underlying regulatory incompatibilities. Here we combine phenotypic data with genome-wide expression data to directly associate aberrant expression patterns with hybrid male sterility between two species of mice. We used a reciprocal cross in which F₁ males are sterile in one direction and fertile in the other direction, allowing us to associate expression differences with sterility rather than with other hybrid phenotypes. We found evidence of extensive over-expression of the X chromosome during spermatogenesis in sterile but not in fertile F₁ hybrid males. Over-expression was most pronounced in genes that are normally expressed after meiosis, consistent with an X chromosome-wide disruption of expression during the later stages of spermatogenesis. This pattern was not a simple consequence of faster evolutionary divergence on the X chromosome, because X-linked expression was highly conserved between the two species. Thus, transcriptional regulation of the X chromosome during spermatogenesis appears particularly sensitive to evolutionary divergence between species. Overall, these data provide evidence for an underlying regulatory basis to reproductive isolation in house mice and underscore the importance of transcriptional regulation of the X chromosome to the evolution of hybrid male sterility.

  2. Gene expression profile in bone marrow and hematopoietic stem cells in mice exposed to inhaled benzene

    Faiola, Brenda; Fuller, Elizabeth S.; Wong, Victoria A.; Recio, Leslie


    Acute myeloid leukemia and chronic lymphocytic leukemia are associated with benzene exposure. In mice, benzene induces chromosomal breaks as a primary mode of genotoxicity in the bone marrow (BM). Benzene-induced DNA lesions can lead to changes in hematopoietic stem cells (HSC) that give rise to leukemic clones. To gain insight into the mechanism of benzene-induced leukemia, we investigated the DNA damage repair and response pathways in total bone marrow and bone marrow fractions enriched for HSC from male 129/SvJ mice exposed to benzene by inhalation. Mice exposed to 100 ppm benzene for 6 h per day, 5 days per week for 2 week showed significant hematotoxicity and genotoxicity compared to air-exposed control mice. Benzene exposure did not alter the level of apoptosis in BM or the percentage of HSC in BM. RNA isolated from total BM cells and the enriched HSC fractions from benzene-exposed and air-exposed mice was used for microarray analysis and quantitative real-time RT-PCR. Interestingly, mRNA levels of DNA repair genes representing distinct repair pathways were largely unaffected by benzene exposure, whereas altered mRNA expression of various apoptosis, cell cycle, and growth control genes was observed in samples from benzene-exposed mice. Differences in gene expression profiles were observed between total BM and HSC. Notably, p21 mRNA was highly induced in BM but was not altered in HSC following benzene exposure. The gene expression pattern suggests that HSC isolated immediately following a 2 weeks exposure to 100 ppm benzene were not actively proliferating. Understanding the toxicogenomic profile of the specific target cell population involved in the development of benzene-associated diseases may lead to a better understanding of the mechanism of benzene-induced leukemia and may identify important interindividual and tissue susceptibility factors.

  3. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane


    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P muscle (65.7 versus 73.8 per cent; P muscle was lower than in the controls (25.9 versus 30.8 per cent; P muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  4. Dietary psyllium fiber increases intestinal heat shock protein 25 expression in mice.

    Ogata, Miyuki; Van Hung, Tran; Tari, Hiroyuki; Arakawa, Teruaki; Suzuki, Takuya


    Heat shock proteins (HSPs) protect intestinal epithelial cell function, integrity and viability against many forms of stress. We hypothesized that dietary fibers (DFs) in the diet may increase HSP expression, since DFs are known to exhibit beneficial effects on intestinal health. The present study investigated the regulation of intestinal HSP expression by DFs, particularly psyllium fiber. Feeding psyllium fiber for 5 d increased HSP25 expression, but not HSP32 and HSP70 expression in the jejunum, ileum, and colon of mice at both the protein and mRNA levels. The increases in HSP25 expression did not correlate with cecal organic acid production by microbial fermentation. The water-insoluble fraction of psyllium fiber largely contributed to the induction of HSP25 expression, but feeding with other water-insoluble DFs from beet, wheat, and oats failed to induce intestinal HSP25 expression. Although the water-holding capacity of psyllium fiber was much higher than those of the other water-insoluble DFs examined, the increase in HSP25 expression induced by feeding polycarbophil, which possesses a high water-holding capacity similar to that of psyllium fiber, was much lower than that induced by psyllium fiber. Finally, induction of malondialdehyde production by hydrogen peroxide, an oxidant, in the colon of mice fed psyllium fiber was lower than that in mice fed with the control diets. Taken together, feeding psyllium fiber, especially the water-insoluble fraction, increases intestinal HSP25 expression and suppresses oxidant-induced malondialdehyde production. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice

    Er-Xia Du; Xiao-Fang Wang; Wu-Chen Yang; Deborah Kaback; Siu-Pok Yee; Chun-Lin Qin; Anne George; Jian-Jun Hao


    Our previous studies have demonstrated that Fam20C promotes differentiation and mineralization of odontoblasts, ameloblasts, osteoblasts and osteocytes during tooth and bone development. Ablation of the Fam20C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice. However, control and regulation of the expression of Fam20C are still unknown. In this study, we generated a transgenic reporter model which expresses green fluorescence protein (GFP) driven by the Fam20C promoter. Recombineering was used to insert a 16 kb fragment of the mouse Fam20C gene (containing the 15 kb promoter and 1.1 kb of exon 1) into a pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence. GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice. Fluorescence was evident in the bone and teeth as early as E17.5. The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth. The expression of GFP was significantly reduced in teeth, alveolar bone and muscle by 8 weeks of age. We also observed colocalization of the GFP signal with the Fam20C antibody in postnatal 1-and 7-day-old animals. Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

  6. Cytokine expression in mice exposed to diesel exhaust particles by inhalation. Role of tumor necrosis factor

    Loft Steffen


    Full Text Available Abstract Background Particulate air pollution has been associated with lung and cardiovascular disease, for which lung inflammation may be a driving mechanism. The pro-inflammatory cytokine, tumor necrosis factor (TNF has been suggested to have a key-role in particle-induced inflammation. We studied the time course of gene expression of inflammatory markers in the lungs of wild type mice and Tnf-/- mice after exposure to diesel exhaust particles (DEPs. Mice were exposed to either a single or multiple doses of DEP by inhalation. We measured the mRNA level of the cytokines Tnf and interleukin-6 (Il-6 and the chemokines, monocyte chemoattractant protein (Mcp-1, macrophage inflammatory protein-2 (Mip-2 and keratinocyte derived chemokine (Kc in the lung tissue at different time points after exposure. Results Tnf mRNA expression levels increased late after DEP-inhalation, whereas the expression levels of Il-6, Mcp-1 and Kc increased early. The expression of Mip-2 was independent of TNF if the dose was above a certain level. The expression levels of the cytokines Kc, Mcp-1 and Il-6, were increased in the absence of TNF. Conclusion Our data demonstrate that Tnf is not important in early DEP induced inflammation and rather exerts negative influence on Mcp-1 and Kc mRNA levels. This suggests that other signalling pathways are important, a candidate being one involving Mcp-1.

  7. Laparotomy in mice induces blood cell expression of inflammatory and stress genes.

    Ko, Fred; Isoda, Fumiko; Mobbs, Charles


    Surgical trauma induces immune and stress responses although its effects on postsurgical inflammatory and stress gene expression remain poorly characterized. This study sought to improve current scientific knowledge by investigating the effects of laparotomy on mouse blood cell inflammatory and stress gene expression. Three-month-old male C57BL/6J mice were subjected to 2% isoflurane or 2% isoflurane with laparotomy and sacrificed 4 h postintervention. Blood was collected and blood cell expression of 158 genes central to inflammatory and stress responses was assayed using quantitative polymerase chain reaction arrays. Mice subjected to isoflurane with laparotomy, compared with mice receiving isoflurane alone, had >2-fold upregulation of genes in inflammation (Osm, IL1rn, IL1b, and Csf1), oxidative stress (Hmox1), heat shock (Hspa1b), growth arrest (Cdkn1a), and DNA repair (Ugt1a2). These genes demonstrated similar expression patterns by Pearson correlation and cluster analysis. Thus, laparotomy induces coordinated, postsurgical blood cell expression of unique inflammatory and stress genes whose roles in influencing surgical outcomes need further investigation.

  8. Adenovirus-mediated eNOS expression augments liver injury after ischemia/reperfusion in mice.

    Arun P Palanisamy

    Full Text Available Hepatic ischemia/reperfusion (l/R injury continues to be a critical problem. The role of nitric oxide in liver I/R injury is still controversial. This study examines the effect of endothelial nitric oxide synthase (eNOS over-expression on hepatic function following I/R. Adenovirus expressing human eNOS (Ad-eNOS was administered by tail vein injection into C57BL/6 mice. Control mice received either adenovirus expressing LacZ or vehicle only. Sixty minutes of total hepatic ischemia was performed 3 days after adenovirus treatment, and mice were sacrificed after 6 or 24 hrs of reperfusion to assess hepatic injury. eNOS over expression caused increased liver injury as evidenced by elevated AST and ALT levels and decreased hepatic ATP content. While necrosis was not pervasive in any group, TUNEL demonstrated significantly increased apoptosis in Ad-eNOS infected livers. Western blotting demonstrated increased levels of protein nitration and upregulation of the pro-apoptotic proteins bax and p53. Our data suggest that over-expression of eNOS is detrimental in the setting of hepatic I/R.

  9. Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice.

    Du, Er-Xia; Wang, Xiao-Fang; Yang, Wu-Chen; Kaback, Deborah; Yee, Siu-Pok; Qin, Chun-Lin; George, Anne; Hao, Jian-Jun


    Our previous studies have demonstrated that Fam20C promotes differentiation and mineralization of odontoblasts, ameloblasts, osteoblasts and osteocytes during tooth and bone development. Ablation of the Fam20C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice. However, control and regulation of the expression of Fam20C are still unknown. In this study, we generated a transgenic reporter model which expresses green fluorescence protein (GFP) driven by the Fam20C promoter. Recombineering was used to insert a 16 kb fragment of the mouse Fam20C gene (containing the 15 kb promoter and 1.1 kb of exon 1) into a pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence. GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice. Fluorescence was evident in the bone and teeth as early as E17.5. The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth. The expression of GFP was significantly reduced in teeth, alveolar bone and muscle by 8 weeks of age. We also observed colocalization of the GFP signal with the Fam20C antibody in postnatal 1- and 7-day-old animals. Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

  10. Gene expression by simian virus 40 large T antigen-induced medulloblastomas in mice

    Xiaoluan Wei; Jie Feng; Yinghe Hu


    Signaling pathways known to have components with mutations in human medulloblastoma include sonic hedgehog, Wnt/beta-catenin and insulin-like growth factor. Microarray analysis was applied to examine the gene expression changes in medulloblastomas of pTet-on/pTRE-SV40Tag transgenic mice. Altogether, 14 112 genes were detectable, including 152 genes with significantly different expression levels. These genes were associated with immunity, the cell cycle, signal transduction, cytoskeleton and metabolism. To further confirm the microarray data, real-time polymerase chain reactions were used to examine the expression changes of genes related to sonic hedgehog, Wnt/beta-catenin and insulin-like growth factor signal pathways. Immunohistochemistry detected insulin receptor substrate-1 in the nuclei of brain tumor tissue cells from pTet-on/pTRE-SV40Tag transgenic mice, suggesting that SV40 large T antigen may activate the insulin-like growth factor signal pathway to promote tumorigenesis.


    González, Betina; González, Candela; Bisagno, Verónica; Urbano, Francisco J.


    Leptin is an adipose-derived hormone that regulates energy balance. Leptin receptors are expressed in extrahypothalamic sites and several reports showed that leptin can influence feeding and locomotor behavior via direct actions on dopaminergic neurons. The leptin deficient mouse (ob/ob) has been used as an animal model of blunted leptin action, and presents with obesity and mild type 2 diabetes. We used ob/ob mice to study the effect of repeated 7-day methamphetamine (METH) administration analyzing locomotion, behavioral sensitization, and somatosensory thalamic mRNA expression of voltage-gated calcium channels and glutamatergic receptors using RT-PCR. We observed reduced METH-mediated responses in ob/ob mice associated with enhanced in mRNA expression of key voltage-gated and glutamate receptors in the somatosensory thalamus. Results described here are important for understanding the control of locomotion and thalamocortical excitability by leptin.

  12. Effect of PAR-2 Deficiency in Mice on KC Expression after Intratracheal LPS Administration

    Julie C. Williams


    Full Text Available Protease activated receptors (PAR have been shown to play a role in inflammation. PAR-2 is expressed by numerous cells in the lung and has either proinflammatory, anti-inflammatory, or no effect depending on the model. Here, we examined the role of PAR-2 in a model of LPS-induced lung inflammation. We found that PAR-2-deficient mice had significantly less KC expression in bronchial lavage fluid compared with wild-type mice but there was no difference in MIP-2 or TNF-α expression. We also found that isolated alveolar and resident peritoneal macrophages lacking PAR-2 showed a similar deficit in KC after LPS stimulation without differences in MIP-2 or TNF-α. Infiltration of neutrophils and macrophages into the lung following LPS administration was not affected by an absence of PAR-2. Our results support the notion that PAR-2 plays a role in LPS activation of TLR4 signaling in macrophages.

  13. Effects of postnatal alcohol exposure on hippocampal gene expression and learning in adult mice.

    Lee, Dong Hoon; Moon, Jihye; Ryu, Jinhyun; Jeong, Joo Yeon; Roh, Gu Seob; Kim, Hyun Joon; Cho, Gyeong Jae; Choi, Wan Sung; Kang, Sang Soo


    Fetal alcohol syndrome (FAS) is a condition resulting from excessive drinking by pregnant women. Symptoms of FAS include abnormal facial features, stunted growth, intellectual deficits and attentional dysfunction. Many studies have investigated FAS, but its underlying mechanisms remain unknown. This study evaluated the relationship between alcohol exposure during the synaptogenesis period in postnatal mice and subsequent cognitive function in adult mice. We delivered two injections, separated by 2 h, of ethanol (3 g/kg, ethanol/saline, 20% v/v) to ICR mice on postnatal day 7. After 10 weeks, we conducted a behavioral test, sacrificed the animals, harvested brain tissue and analyzed hippocampal gene expression using a microarray. In ethanol-treated mice, there was a reduction in brain size and decreased neuronal cell number in the cortex, and also cognitive impairment. cDNA microarray results indicated that 1,548 genes showed a > 2-fold decrease in expression relative to control, whereas 974 genes showed a > 2-fold increase in expression relative to control. Many of these genes were related to signal transduction, synaptogenesis and cell membrane formation, which are highlighted in our findings.

  14. Th1/Th2 cytokines' expression and production by propolis-treated mice.

    Orsatti, Cláudio Lera; Missima, Fabiane; Pagliarone, Ana Carolina; Sforcin, José Maurício


    Propolis is a natural product extensively used in food and beverages to improve health and to prevent diseases, showing immunomodulatory properties. The goal of this work was to evaluate the effect of propolis administration over a short-term to mice on Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-10) cytokines' expression and production. Propolis was administered for 3 days to mice by gavage, spleens were removed and RNA was extracted to assess cytokines' expression by real-time PCR. Supernatants of spleen cell cultures were used for cytokines determination by ELISA. Propolis administration to mice did not affect IL-2, IL-4 and IL-10 expression and production, while IFN-gamma production was inhibited in the splenocyte cultures stimulated or not by Con A. Since IFN-gamma is a pro-inflammatory cytokine, our data suggest that propolis administration over a short-term to mice may be associated with anti-inflammatory effects in vivo, and further assays could check propolis efficiency in inflammatory diseases. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  15. Endurance training inhibits insulin clearance and IDE expression in Swiss mice.

    José M Costa-Júnior

    Full Text Available Endurance training improves peripheral insulin sensitivity in the liver and the skeletal muscle, but the mechanism for this effect is poorly understood. Recently, it was proposed that insulin clearance plays a major role in both glucose homeostasis and insulin sensitivity. Therefore, our goal was to determine the mechanism by which endurance training improves insulin sensitivity and how it regulates insulin clearance in mice.Mice were treadmill-trained for 4 weeks at 70-80% of maximal oxygen consumption (VO2 max for 60 min, 5 days a week. The glucose tolerance and the insulin resistance were determined using an IPGTT and an IPITT, respectively, and the insulin decay rate was calculated from the insulin clearance. Protein expression and phosphorylation in the liver and the skeletal muscle were ascertained by Western blot.Trained mice exhibited an increased VO2 max, time to exhaustion, glucose tolerance and insulin sensitivity. They had smaller fat pads and lower plasma concentrations of insulin and glucose. Endurance training inhibited insulin clearance and reduced expression of IDE in the liver, while also inhibiting insulin secretion by pancreatic islets. There was increased phosphorylation of both the canonical (IR-AKT and the non-canonical (CaMKII-AMPK-ACC insulin pathways in the liver of trained mice, whereas only the CaMKII-AMPK pathway was increased in the skeletal muscle.Endurance training improved glucose homeostasis not only by increasing peripheral insulin sensitivity but also by decreasing insulin clearance and reducing IDE expression in the liver.

  16. Genipin inhibits mitochondrial uncoupling protein 2 expression and ameliorates podocyte injury in diabetic mice.

    Wenjing Qiu

    Full Text Available Diabetic nephropathy (DN is one of the most common causes of end stage renal disease (ESRD in China, which requires renal replacement therapy. Recent investigations have suggested an essential role of podocyte injury in the initial stage of DN. This study investigated the potential therapeutic role of genipin, an active extract from a traditional Chinese medicine, on progression of DN in diabetic mice induced by intraperitoneally injection of streptozocin (STZ. In diabetic mice, orally administration of genipin postponed the progression of DN, as demonstrated by ameliorating body weight loss and urine albumin leakage, attenuating glomerular basement membrane thickness, restoring the podocyte expression of podocin and WT1 in diabetic mice. The protective role of genipin on DN is probably through suppressing the up-regulation of mitochondrial uncoupling protein 2 (UCP2 in diabetic kidneys. Meanwhile, through inhibiting the up-regulation of UCP2, genipin restores podocin and WT1 expression in cultured podocytes and attenuates glucose-induced albumin leakage through podocytes monolayer. Therefore, these results revealed that genipin inhibited UCP2 expression and ameliorated podocyte injury in DN mice.

  17. Expression of NALP3 in the Spleen of Mice with Portal Hypertension

    夏泽锋; 王国斌; 万赤丹; 刘涛; 王帅; 王博; 程锐


    This study examined the mRNA expression of NALP3 in the spleen of the mice with hypersplenism due to portal hypertension(PH).The mouse hypersplenism models were established by oral administration of tetrachloromethane(2 mL/kg/week for 12 weeks by oral gavage).All the mice were randomly divided into a control group and an experimental group.The blood routine test was conducted,spleen index was calculated and spleen was histologically examined.Portal vein sera were taken for detection of the level of uric aci...

  18. Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment.

    Aleksunes, Lauren M; Augustine, Lisa M; Scheffer, George L; Cherrington, Nathan J; Manautou, José E


    The goal of this study was to identify alterations in mRNA and protein expression of various xenobiotic transport proteins in mouse kidney during cisplatin-induced acute renal failure. For this purpose, male C57BL/6J mice received a single dose of cisplatin (18 mg/kg, i.p.) or vehicle. Four days later, tissues were collected for assessment of plasma BUN, histopathological analysis of renal lesions, and mRNA and Western blot analysis of renal transporters including organic anion and cation transporters (Oat, Oct), organic anion transporting polypeptides (Oatp), multidrug resistance-associated proteins (Mrp), multidrug resistance proteins (Mdr), breast cancer resistance protein (Bcrp) and multidrug and toxin extrusion proteins (Mate). Cisplatin treatment caused necrosis of renal proximal tubules along with elevated plasma BUN and renal kidney injury molecule-1 mRNA expression. Cisplatin-induced renal injury increased mRNA and protein levels of the efflux transporters Mrp2, Mrp4, Mrp5, Mdr1a and Mdr1b. Uptake transporters Oatp2a1 and Oatp2b1 mRNA were also up-regulated following cisplatin. By contrast, expression of Oat1, Oat2, Oct2 and Oatp1a1 mRNA was reduced in cisplatin-treated mice. Expression of several uptake and efflux transporters was unchanged in cisplatin-treated mice. Apical staining of Mrp2 and Mrp4 proteins was enhanced in proximal tubules from cisplatin-treated mice. Collectively, these expression patterns suggest coordinated regulation of uptake and efflux pathways during cisplatin-induced renal injury. Reduced expression of basolateral and apical uptake transporters along with enhanced transcription of export transporters likely represents an adaptation to lower intracellular accumulation of chemicals, prevent their reabsorption and enhance urinary clearance.

  19. Heterogeneous Nuclear Ribonucleoprotein F Stimulates Sirtuin-1 Gene Expression and Attenuates Nephropathy Progression in Diabetic Mice.

    Lo, Chao-Sheng; Shi, Yixuan; Chenier, Isabelle; Ghosh, Anindya; Wu, Chin-Han; Cailhier, Jean-Francois; Ethier, Jean; Lattouf, Jean-Baptiste; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao-Ling; Chan, John S D


    We investigated the mechanism of heterogeneous nuclear ribonucleoprotein F (hnRNP F) renoprotective action in a type 2 diabetes (T2D) mouse model (db/db). Immortalized rat renal proximal tubular cells (IRPTCs) and kidneys from humans with T2D were also studied. The db/db mice developed hyperglycemia, oxidative stress, and nephropathy at age 20 weeks compared with their db/m littermates. These abnormalities, with the exception of hyperglycemia, were attenuated in db/dbhnRNP F-transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs. Sirtuin-1, Foxo3α, and catalase expression were significantly decreased in RPTCs from db/db mice and normalized in db/dbhnRNP F-Tg mice. In vitro, hnRNP F overexpression stimulated Sirtuin-1 and Foxo3α with downregulation of acetylated p53 expression and prevented downregulation of Sirtuin-1 and Foxo3α expression in IRPTCs by high glucose plus palmitate. Transfection of Sirtuin-1 small interfering RNA prevented hnRNP F stimulation of Foxo3α and downregulation of acetylated p53 expression. hnRNP F stimulated Sirtuin-1 transcription via hnRNP F-responsive element in the Sirtuin-1 promoter. Human T2D kidneys exhibited more RPTC apoptosis and lower expression of hnRNP F, SIRTUIN-1, and FOXO3α than nondiabetic kidneys. Our results demonstrate that hnRNP F protects kidneys against oxidative stress and nephropathy via stimulation of Sirtuin-1 expression and signaling in diabetes. © 2017 by the American Diabetes Association.

  20. Dietary glutamine supplementation modulates Th1/Th2 cytokine and interleukin-6 expressions in septic mice.

    Yeh, Chiu Li; Hsu, Chun-Sun; Yeh, Sung-Ling; Chen, Wei-Jao


    Glutamine (Gln) has been demonstrated to have benefit in the modulation of systemic immunity in sepsis. However, the effects of Gln on local immunity and intra-lymphocyte cytokine expression have not been investigated in mice with gut-derived sepsis. This study evaluated the influence of a Gln-enriched diet on interleukin (IL)-6 expression in organs and Th1/Th2 type cytokine production within lymphocytes in septic mice. Male ICR mice were assigned to control and Gln groups. The control group was fed a semi-purified diet, while in the Gln group, Gln replaced part of the casein. After feeding the respective diets for 3 weeks, sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0, 6, 12 and 24h after CLP and their organs were harvested for further analysis. Results showed that IL-6 levels in the liver were decreased, whereas levels were increased in the lungs, kidneys and intestines with the progression of sepsis in both groups. Also, intra-lymphocyte interferon (IFN)-gamma expression decreased and IL-4 expression increased during sepsis. Compared to the control group, the Gln group had higher levels of IL-6 in the liver and lower levels in other organs at various time points. Lymphocyte IFN-gamma expression in the Gln group was higher, and IL-4 levels were lower than those of the control group after CLP. These results suggest that Gln supplementation decreased IL-6 production in non-hepatic organs, while reducing intra-lymphocyte IL-4 and enhancing IFN-gamma expressions. This change may reverse the Th2 type response to a more-balanced Th1/Th2 response during sepsis.

  1. Excess of leptin inhibits hypothalamic KiSS-1 expression in pubertal mice

    Sung Yeon Ahn


    Full Text Available &lt;B&gt;Purpose:&lt;/B&gt; Leptin has been considered a link between metabolic state and reproductive activity. Defective reproductive function can occur in leptin-deficient and leptin-excessive conditions. The aim of this study was to examine the effects of centrally injected leptin on the hypothalamic &lt;I&gt;KiSS-1&lt;/I&gt; system in relation to gonadotropin-releasing hormone (GnRH action in the initial stage of puberty. &lt;B&gt;Methods:&lt;/B&gt; Leptin (1 μg was injected directly into the ventricle of pubertal female mice. The resultant gene expressions of hypothalamic &lt;I&gt;GnRH&lt;/I&gt; and &lt;I&gt;KiSS-1&lt;/I&gt; and pituitary LH, 2 and 4 hours after injection, were compared with those of saline-injected control mice. The changes in the gene expressions after blocking the GnRH action were also analyzed. &lt;B&gt;Results:&lt;/B&gt; The basal expression levels of &lt;I&gt;KiSS-1&lt;/I&gt;, GnRH, and LH were significantly higher in the pubertal mice than in the prepubertal mice. The 1-μg leptin dose significantly decreased the mRNA expression levels of &lt;I&gt;KiSS-1&lt;/I&gt;, GnRH, and LH in the pubertal mice. A &lt;I&gt;GnRH&lt;/I&gt; antagonist significantly increased the &lt;I&gt;KiSS-1&lt;/I&gt; and GnRH mRNA expression levels, and the additional leptin injection decreased the gene expression levels compared with those in the control group. &lt;B&gt;Conclusion:&lt;/B&gt; The excess leptin might have suppressed the central reproductive axis in the pubertal mice by inhibiting the &lt;I&gt;KiSS-1&lt;/I&gt; expression, and this mechanism is independent of the GnRH-LHestradiol feedback loop.

  2. Dietary fish oil regulates gene expression of cholesterol and bile acid transporters in mice.

    Kamisako, Toshinori; Tanaka, Yuji; Ikeda, Takanori; Yamamoto, Kazuo; Ogawa, Hiroshi


      Fish oil rich in n-3 polyunsaturated fatty acids is known to affect hepatic lipid metabolism. Several studies have demonstrated that fish oil may affect the bile acid metabolism as well as lipid metabolism, whereas only scarce data are available. The aim of this study was to investigate the effect of fish oil on the gene expression of the transporters and enzymes related to bile acid as well as lipid metabolism in the liver and small intestine.   Seven-week old male C57BL/6 mice were fed diets enriched in 10% soybean oil or 10% fish oil for 4 weeks. After 4 weeks, blood, liver and small intestine were obtained.   Hepatic mRNA expression of lipids (Abcg5/8, multidrug resistance gene product 2) and bile acids transporters (bile salt export pump, multidrug resistance associated protein 2 and 3, organic solute transporter α) was induced in fish oil-fed mice. Hepatic Cyp8b1, Cyp27a1 and bile acid CoA : amino acid N-acyltransferase were increased in fish oil-fed mice compared with soybean-oil fed mice. Besides, intestinal cholesterol (Abcg5/8) and bile acid transporters (multidrug resistance associated protein 2 and organic solute transporter α) were induced in fish oil-fed mice.   Fish oil induced the expression of cholesterol and bile acid transporters not only in liver but in intestine. The upregulation of Abcg5/g8 by fish oil is caused by an increase in cellular 27-HOC through Cyp27a1 induction. The hepatic induction of bile acid synthesis through Cyp27a1 may upregulate expression of bile acid transporters in both organs. © 2012 The Japan Society of Hepatology.

  3. Quercetin prevents progression of disease in elastase/LPS-exposed mice by negatively regulating MMP expression

    Martinez Fernando J


    Full Text Available Abstract Background Chronic obstructive pulmonary disease (COPD is characterized by chronic bronchitis, emphysema and irreversible airflow limitation. These changes are thought to be due to oxidative stress and an imbalance of proteases and antiproteases. Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. We hypothesized that quercetin reduces lung inflammation and improves lung function in elastase/lipopolysaccharide (LPS-exposed mice which show typical features of COPD, including airways inflammation, goblet cell metaplasia, and emphysema. Methods Mice treated with elastase and LPS once a week for 4 weeks were subsequently administered 0.5 mg of quercetin dihydrate or 50% propylene glycol (vehicle by gavage for 10 days. Lungs were examined for elastance, oxidative stress, inflammation, and matrix metalloproteinase (MMP activity. Effects of quercetin on MMP transcription and activity were examined in LPS-exposed murine macrophages. Results Quercetin-treated, elastase/LPS-exposed mice showed improved elastic recoil and decreased alveolar chord length compared to vehicle-treated controls. Quercetin-treated mice showed decreased levels of thiobarbituric acid reactive substances, a measure of lipid peroxidation caused by oxidative stress. Quercetin also reduced lung inflammation, goblet cell metaplasia, and mRNA expression of pro-inflammatory cytokines and muc5AC. Quercetin treatment decreased the expression and activity of MMP9 and MMP12 in vivo and in vitro, while increasing expression of the histone deacetylase Sirt-1 and suppressing MMP promoter H4 acetylation. Finally, co-treatment with the Sirt-1 inhibitor sirtinol blocked the effects of quercetin on the lung phenotype. Conclusions Quercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.

  4. Aged mice have increased inflammatory monocyte concentration and altered expression of cell-surface functional receptors

    Kelley Strohacker; Whitney L Breslin; Katie C Carpenter; Brian K McFarlin


    The expression of monocyte cell-surface receptors represents one index of immune dysfunction, which is common with aging. Although mouse models of aging are prevalent, monocyte subset assessment is rare. Our purpose was to compare cell receptor expression on classic (CD115+/Gr-1high) and non-classic (CD115+/Gr-1low) monocytes from 80- or 20-week-old CD-1 mice. Three-colour flow cytometry was used to determine the concentration of monocyte subsets and their respective cell-surface expression of TLR2, TLR4, CD80, CD86, MHC II and CD54. These receptors were selected because they have been previously associated with altered monocyte function. Data were analysed with independent -tests; significance was set at < 0.05. Old mice had a greater concentration of both classic (258%, =0.003) and non-classic (70%, =0.026) monocytes. The classic : non-classic monocyte ratio doubled in old as compared with that in young mice (=0.006), indicating a pro-inflammatory shift. TLR4 ($\\downarrow$27%, =0.001) and CD80 ($\\downarrow$37%, =0.004) were decreased on classic monocytes from old as compared with those from young mice. TLR2 ($\\uparrow$24%, =0.002) and MHCII ($\\downarrow$21%, =0.026) were altered on non-classic monocytes from old as compared with those from young mice. The increased classic : non-classic monocyte ratio combined with changes in the cell-surface receptor expression on both monocyte subsets is indicative of immune dysfunction, which may increase age-associated disease risk.

  5. Therapeutic potential of amniotic-fluid-derived stem cells on liver fibrosis model in mice

    Shao-Yu Peng


    Conclusion: The possible repair mechanism from our data revealed that EGFP-mAFSCs may fuse with the recipient liver cells. Overall, EGFP-mAFSCs can ameliorate liver fibrosis in mice, thus providing insight into the future development of regenerative medicine.

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

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


    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.

  7. Altered Expression of Somatostatin Receptors in Pancreatic Islets from NOD Mice Cultured at Different Glucose Concentrations In Vitro and in Islets Transplanted to Diabetic NOD Mice In Vivo

    Eva Ludvigsen


    Full Text Available Somatostatin acts via five receptors (sst1-5. We investigated if the changes in pancreatic islet sst expression in diabetic NOD mice compared to normoglycemic mice are a consequence of hyperglycemia or the ongoing immune reaction in the pancreas. Pancreatic islets were isolated from NOD mice precultured for 5 days and further cultured for 3 days at high or low glucose before examined. Islets were also isolated from NOD mice and transplanted to normal or diabetic mice in a number not sufficient to cure hyperglycemia. After three days, the transplants were removed and stained for sst1-5 and islet hormones. Overall, changes in sst islet cell expression were more common in islets cultured in high glucose concentration in vitro as compared to the islet transplantation in vivo to diabetic mice. The beta and PP cells exhibited more frequent changes in sst expression, while the alpha and delta cells were relatively unaffected by the high glucose condition. Our findings suggest that the glucose level may alter sst expressed in islets cells; however, immune mechanisms may counteract such changes in islet sst expression.

  8. Increased stathmin1 expression in the dentate gyrus of mice causes abnormal axonal arborizations.

    Kohei Yamada

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is involved in multiple brain functions. To clarify the cause of abnormal behavior in PACAP deficient-mice, we attempted the identification of genes whose expression was altered in the dentate gyrus of PACAP-deficient mice using the differential display method. Expression of stathmin1 was up-regulated in the dentate gyrus at both the mRNA and protein levels. PACAP stimulation inhibited stathmin1 expression in PC12 cells, while increased stathmin1expression in neurons of the subgranular zone and in primary cultured hippocampal neurons induced abnormal arborization of axons. We also investigated the pathways involved in PACAP deficiency. Ascl1 binds to E10 box of the stathmin1 promoter and increases stathmin1 expression. Inhibitory bHLH proteins (Hes1 and Id3 were rapidly up-regulated by PACAP stimulation, and Hes1 could suppress Ascl1 expression and Id3 could inhibit Ascl1 signaling. We also detected an increase of stathmin1 expression in the brains of schizophrenic patients. These results suggest that up-regulation of stathmin1 in the dentate gyrus, secondary to PACAP deficiency, may create abnormal neuronal circuits that cause abnormal behavior.

  9. Myostatin expression, lymphocyte population, and potential cytokine production correlate with predisposition to high-fat diet induced obesity in mice.

    Jeri-Anne Lyons

    Full Text Available A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO resistant (SWR/J and susceptible (C57BL/6 mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4(+/CD44(hi and CD8(+/CD44(hi cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.

  10. Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

    Kazuaki Miyagawa

    Full Text Available Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH, Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

  11. Use of the viral 2A peptide for bicistronic expression in transgenic mice

    Trichas Georgios


    Full Text Available Abstract Background Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner. Results To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato and a nuclear localised green fluorescent protein (H2B-GFP, separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating

  12. Expression of complement system components during aging and amyloid deposition in APP transgenic mice

    Wiederhold Karl-Heinz


    Full Text Available Abstract Background A causal role of the complement system in Alzheimer's disease pathogenesis has been postulated based on the identification of different activated components up to the membrane attack complex at amyloid plaques in brain. However, histological studies of amyloid plaque bearing APP transgenic mice provided only evidence for an activation of the early parts of the complement cascade. To better understand the contribution of normal aging and amyloid deposition to the increase in complement activation we performed a detailed characterization of the expression of the major mouse complement components. Methods APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 mice were used at different ages. mRNA was quantified by Realtime PCR and the age- as well as amyloid induced changes determined. The protein levels of complement C1q and C3 were analysed by Western blotting. Histology was done to test for amyloid plaque association and activation of the complement cascade. Results High mRNA levels were detected for C1q and some inhibitory complement components. The expression of most activating components starting at C3 was low. Expression of C1q, C3, C4, C5 and factor B mRNA increased with age in control C57BL/6 mice. C1q and C3 mRNA showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was confirmed on the protein level using Western blotting, whereas immunohistology indicated a recruitment of complement to amyloid plaques up to the C3 convertase. Conclusion Early but not late components of the mouse complement system show an age-dependent increase in expression. The response to amyloid deposition is comparatively smaller. The low expression of C3 and C5 and failure to upregulate C5 and downstream components differs from human AD brain and likely contributes to the lack of full complement activation in APP transgenic mice.

  13. Shuanghuanglian injection downregulates nuclear factor-kappa B expression in mice with viral encephalitis

    Naibing Gu; Ye Tian; Zhengli Di; Caiping Han; Hui Lei; Gejuan Zhang


    A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension.Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups.Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis.After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days,intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased.To elucidate the relationship between intelectin-2 and nuclear factor-kappa B,mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA.Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased.Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production,thus inhibiting inflammation associated with viral encephalitis.

  14. Caveolin-1, caveolin-3 and VEGF expression in the masticatory muscles of mdx mice

    Alexander Spassov


    Full Text Available Duchenne muscular dystrophy (DMD and murine X-linked muscular dystrophy (mdx, its murine model, are characterized by muscle damage and muscle weakness associated with inflammation and new vessel formation. Caveolins, dystrophin-associated proteins, are involved in the pathogenesis of DMD, because increased numbers of caveolae are found in DMD and mdx hindlimb muscles. Caveolae influence angiogenesis due to their content of vascular endothelial growth factor (VEGF receptors. Orofacial muscles in mdx mice undergo muscle necrosis followed by muscle regeneration. To ascertain the role of caveolins and VEGF in the pathogenesis of dystrophic masticatory muscles, we examined the expression of caveolin-1 (cav-1, caveolin-3 (cav-3 and VEGF in control and mdx mice. In mdx masticatory muscles, no changes in transcript and protein levels of VEGF were found, whereas cav-1 and cav-3 expression was increased. Using immunohistochemistry, a strong sarcolemmal staining of caveolin-3 in regenerated muscle fibers was found. Furthermore, immunohistochemistry with the caveolin-1 antibody showed an increase in the amount of blood vessels in areas with regenerating muscle fibers. Dystrophic masticatory muscles showed changes comparable to those of hindlimb muscles in the expression of cav-1 and cav-3. The angiogenesis seems to be unaffected in the jaw muscles of mdx mice. We speculate that the increased caveolin expression could cause extensive and efficient muscle regeneration. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 2, pp. 291–298

  15. Leptin modulated changes in adipose tissue protein expression in ob/ob mice.

    Zhang, Wei; Ambati, Suresh; Della-Fera, Mary Anne; Choi, Yang-Ho; Baile, Clifton A; Andacht, Tracy M


    Comparative proteomic analyses were performed in adipose tissue of leptin-deficient ob/ob mice treated with leptin or control buffer in order to identify the protein expression changes as the potential targets of leptin. Mice were treated with either phosphate-buffered saline (control) or 10 µg/day leptin for 14 days via subcutaneous osmotic minipumps. Total protein from white adipose tissue was extracted and labeled with different fluorescent cyanine dyes for analysis by two-dimensional difference gel electrophoresis (DIGE). Spots that were differentially expressed and appeared to have sufficient material for mass spectrometry analysis were picked and digested with trypsin and subjected to MALDI-TOF MS for protein identification. Twelve functional protein groups were found differentially expressed in adipose tissue of leptin-treated vs. control ob/ob mice, including molecular chaperones and redox proteins such as calreticulin (CALR), protein disulfide isomerase-associated 3 (PDIA3), prohibitin (PHB), and peroxiredoxin-6 (PRDX6); cytoskeleton proteins such as β actin, desmin, and α-tubulin; and some other proteins. The mRNA levels of CALR, PDIA3, and PHB were measured by real-time reverse transcription-PCR and found to be upregulated (P leptin's effects on lipid metabolism and apoptosis may be mediated in part by alterations in expression of molecular chaperones and redox proteins for regulating endoplasmic reticulum stress and cytoskeleton proteins for regulating mitochondrial morphology.

  16. Gene Expression Profile in the Liver of BALB/c Mice Infected with Fasciola hepatica.

    Rojas-Caraballo, Jose; López-Abán, Julio; Fernández-Soto, Pedro; Vicente, Belén; Collía, Francisco; Muro, Antonio


    Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology. A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However

  17. Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice

    Weber Mitch


    Full Text Available Abstract Background Women with polycystic ovary syndrome (PCOS are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD, which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a mice, possessing a mutation (Ay in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction. Methods Female LY mice received daily oral doses of either 0.01 mg pioglitazone (n = 4 or an equal volume of vehicle (DMSO; n = 4 for 8 weeks. At the end of treatment, ovaries were removed and DNA microarrays were used to analyze differential gene expression. Results Twenty-seven genes showed at least a two-fold difference in ovarian expression with pioglitazone treatment. These included leptin, angiopoietin, angiopoietin-like 4, Foxa3, PGE1 receptor, resistin-like molecule-alpha (RELM, and actin-related protein 6 homolog (ARP6. For most altered genes, pioglitazone changed levels of expression to those seen in untreated C57BL/6J(a/a non-mutant lean mice. Conclusion TZD administration may influence ovarian function via numerous diverse mechanisms that may or may not be directly related to insulin/IGF signaling.

  18. Decrease in expression of maternal effect gene Mater is associated with maternal ageing in mice.

    Lu, Yong-qing; He, Xie-chao; Zheng, Ping


    What factors in mouse oocytes are involved in the ageing-related decline in oocyte quality? The maternal effect gene Mater is involved in ageing-related oocyte quality decline in mice. Premature loss of centromere cohesion is a hallmark of ageing-related oocyte quality decline; the maternal effect gene Mater (maternal antigen that embryos require, also known as Nlrp5) is required for preimplantation embryo development beyond the 2-cell stage, and mRNA expression of Mater decreases with maternal ageing. Mater protein expression level in mature oocytes from 7 young (5-8 weeks old) to 7 old mice (41-68 weeks old) was compared by immunoblotting analysis. Wild-type and Mater-null mice were used to examine whether Mater is necessary for maintaining normal centromere cohesion by means of cytogenetic karyotyping, time-lapse confocal microscopy and immunofluorescence staining. Mater protein is decreased in mature oocytes from old versus young mice (P = 0.0022). Depletion of Mater from oocytes leads to a reduction in centromere cohesion, manifested by precocious sister chromatid separation, enlargement of sister centromere distance and misalignment of chromosomes in the metaphase plate during meiosis I and II. This study was conducted in mice. Whether or not the results are applicable to human remains further elucidation. In addition, we were unable to confirm if the strain of mice (C57BL/6XSv129) at the age of 41-68 weeks old has the 'cohesin-loss' phenotype. Investigating Mater's functional mechanisms could provide fresh insights into understanding how the ageing-related oocyte quality decline occurs. N/A. This work was supported by the research grant from Chinese NSFC to P.Z. (31071274). We have no conflict of interests to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:

  19. Tissue-specific expression of monocarboxylate transporters during fasting in mice.

    Schutkowski, Alexandra; Wege, Nicole; Stangl, Gabriele I; König, Bettina


    Monocarboxylates such as pyruvate, lactate and ketone bodies are crucial for energy supply of all tissues, especially during energy restriction. The transport of monocarboxylates across the plasma membrane of cells is mediated by monocarboxylate transporters (MCTs). Out of 14 known mammalian MCTs, six isoforms have been functionally characterized to transport monocarboxylates and short chain fatty acids (MCT1-4), thyroid hormones (MCT8, -10) and aromatic amino acids (MCT10). Knowledge on the regulation of the different MCT isoforms is rare. In an attempt to get more insights in regulation of MCT expression upon energy deprivation, we carried out a comprehensive analysis of tissue specific expression of five MCT isoforms upon 48 h of fasting in mice. Due to the crucial role of peroxisome proliferator-activated receptor (PPAR)-α as a central regulator of energy metabolism and as known regulator of MCT1 expression, we included both wildtype (WT) and PPARα knockout (KO) mice in our study. Liver, kidney, heart, small intestine, hypothalamus, pituitary gland and thyroid gland of the mice were analyzed. Here we show that the expression of all examined MCT isoforms was markedly altered by fasting compared to feeding. Expression of MCT1, MCT2 and MCT10 was either increased or decreased by fasting dependent on the analyzed tissue. MCT4 and MCT8 were down-regulated by fasting in all examined tissues. However, PPARα appeared to have a minor impact on MCT isoform regulation. Due to the fundamental role of MCTs in transport of energy providing metabolites and hormones involved in the regulation of energy homeostasis, we assumed that the observed fasting-induced adaptations of MCT expression seem to ensure an adequate energy supply of tissues during the fasting state. Since, MCT isoforms 1-4 are also necessary for the cellular uptake of drugs, the fasting-induced modifications of MCT expression have to be considered in future clinical care algorithms.

  20. Tissue-specific expression of monocarboxylate transporters during fasting in mice.

    Alexandra Schutkowski

    Full Text Available Monocarboxylates such as pyruvate, lactate and ketone bodies are crucial for energy supply of all tissues, especially during energy restriction. The transport of monocarboxylates across the plasma membrane of cells is mediated by monocarboxylate transporters (MCTs. Out of 14 known mammalian MCTs, six isoforms have been functionally characterized to transport monocarboxylates and short chain fatty acids (MCT1-4, thyroid hormones (MCT8, -10 and aromatic amino acids (MCT10. Knowledge on the regulation of the different MCT isoforms is rare. In an attempt to get more insights in regulation of MCT expression upon energy deprivation, we carried out a comprehensive analysis of tissue specific expression of five MCT isoforms upon 48 h of fasting in mice. Due to the crucial role of peroxisome proliferator-activated receptor (PPAR-α as a central regulator of energy metabolism and as known regulator of MCT1 expression, we included both wildtype (WT and PPARα knockout (KO mice in our study. Liver, kidney, heart, small intestine, hypothalamus, pituitary gland and thyroid gland of the mice were analyzed. Here we show that the expression of all examined MCT isoforms was markedly altered by fasting compared to feeding. Expression of MCT1, MCT2 and MCT10 was either increased or decreased by fasting dependent on the analyzed tissue. MCT4 and MCT8 were down-regulated by fasting in all examined tissues. However, PPARα appeared to have a minor impact on MCT isoform regulation. Due to the fundamental role of MCTs in transport of energy providing metabolites and hormones involved in the regulation of energy homeostasis, we assumed that the observed fasting-induced adaptations of MCT expression seem to ensure an adequate energy supply of tissues during the fasting state. Since, MCT isoforms 1-4 are also necessary for the cellular uptake of drugs, the fasting-induced modifications of MCT expression have to be considered in future clinical care algorithms.

  1. Tissue-Specific Expression of Monocarboxylate Transporters during Fasting in Mice

    Schutkowski, Alexandra; Wege, Nicole; Stangl, Gabriele I.; König, Bettina


    Monocarboxylates such as pyruvate, lactate and ketone bodies are crucial for energy supply of all tissues, especially during energy restriction. The transport of monocarboxylates across the plasma membrane of cells is mediated by monocarboxylate transporters (MCTs). Out of 14 known mammalian MCTs, six isoforms have been functionally characterized to transport monocarboxylates and short chain fatty acids (MCT1-4), thyroid hormones (MCT8, -10) and aromatic amino acids (MCT10). Knowledge on the regulation of the different MCT isoforms is rare. In an attempt to get more insights in regulation of MCT expression upon energy deprivation, we carried out a comprehensive analysis of tissue specific expression of five MCT isoforms upon 48 h of fasting in mice. Due to the crucial role of peroxisome proliferator-activated receptor (PPAR)-α as a central regulator of energy metabolism and as known regulator of MCT1 expression, we included both wildtype (WT) and PPARα knockout (KO) mice in our study. Liver, kidney, heart, small intestine, hypothalamus, pituitary gland and thyroid gland of the mice were analyzed. Here we show that the expression of all examined MCT isoforms was markedly altered by fasting compared to feeding. Expression of MCT1, MCT2 and MCT10 was either increased or decreased by fasting dependent on the analyzed tissue. MCT4 and MCT8 were down-regulated by fasting in all examined tissues. However, PPARα appeared to have a minor impact on MCT isoform regulation. Due to the fundamental role of MCTs in transport of energy providing metabolites and hormones involved in the regulation of energy homeostasis, we assumed that the observed fasting-induced adaptations of MCT expression seem to ensure an adequate energy supply of tissues during the fasting state. Since, MCT isoforms 1–4 are also necessary for the cellular uptake of drugs, the fasting-induced modifications of MCT expression have to be considered in future clinical care algorithms. PMID:25390336

  2. Generation and Characterization of Mice Expressing a Conditional Allele of the Interleukin-1 Receptor Type 1

    Robson, Matthew J.; Zhu, Chong-Bin; Quinlan, Meagan A.; Botschner, David A.; Baganz, Nicole L.; Lindler, Kathryn M.; Thome, Jason G.; Hewlett, William A.; Blakely, Randy D.


    The cytokines IL-1α and IL-1β exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1). Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP), with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV)-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development. PMID:26930558

  3. Generation and Characterization of Mice Expressing a Conditional Allele of the Interleukin-1 Receptor Type 1.

    Matthew J Robson

    Full Text Available The cytokines IL-1α and IL-1β exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1. Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP, with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development.

  4. SCD1 Expression is dispensable for hepatocarcinogenesis induced by AKT and Ras oncogenes in mice.

    Lei Li

    Full Text Available Increased de novo lipogenesis is one of the major metabolic events in cancer. In human hepatocellular carcinoma (HCC, de novo lipogenesis has been found to be increased and associated with the activation of AKT/mTOR signaling. In mice, overexpression of an activated form of AKT results in increased lipogenesis and hepatic steatosis, ultimately leading to liver tumor development. Hepatocarcinogenesis is dramatically accelerated when AKT is co-expressed with an oncogenic form of N-Ras. SCD1, the major isoform of stearoyl-CoA desaturases, catalyzing the conversion of saturated fatty acids (SFA into monounsaturated fatty acids (MUFA, is a key enzyme involved in de novo lipogenesis. While many studies demonstrated the requirement of SCD1 for tumor cell growth in vitro, whether SCD1 is necessary for tumor development in vivo has not been previously investigated. Here, we show that genetic ablation of SCD1 neither inhibits lipogenesis and hepatic steatosis in AKT-overexpressing mice nor affects liver tumor development in mice co-expressing AKT and Ras oncogenes. Molecular analysis showed that SCD2 was strongly upregulated in liver tumors from AKT/Ras injected SCD1(-/- mice. Noticeably, concomitant silencing of SCD1 and SCD2 genes was highly detrimental for the growth of AKT/Ras cells in vitro. Altogether, our study provides the evidence, for the first time, that SCD1 expression is dispensable for AKT/mTOR-dependent hepatic steatosis and AKT/Ras-induced hepatocarcinogenesis in mice. Complete inhibition of stearoyl-CoA desaturase activity may be required to efficiently suppress liver tumor development.

  5. Long tract of untranslated CAG repeats is deleterious in transgenic mice.

    Ren-Jun Hsu

    Full Text Available The most frequent trinucleotide repeat found in human disorders is the CAG sequence. Expansion of CAG repeats is mostly found in coding regions and is thought to cause diseases through a protein mechanism. Recently, expanded CAG repeats were shown to induce toxicity at the RNA level in Drosophila and C. elegans. These findings raise the possibility that CAG repeats may trigger RNA-mediated pathogenesis in mammals. Here, we demonstrate that transgenic mice expressing EGFP transcripts with long CAG repeats in the 3' untranslated region develop pathogenic features. Expression of the transgene was directed to the muscle in order to compare the resulting phenotype to that caused by the CUG expansion, as occurs in myotonic dystrophy. Transgenic mice expressing 200, but not those expressing 0 or 23 CAG repeats, showed alterations in muscle morphology, histochemistry and electrophysiology, as well as abnormal behavioral phenotypes. Expression of the expanded CAG repeats in testes resulted in reduced fertility due to defective sperm motility. The production of EGFP protein was significantly reduced by the 200 CAG repeats, and no polyglutamine-containing product was detected, which argues against a protein mechanism. Moreover, nuclear RNA foci were detected for the long CAG repeats. These data support the notion that expanded CAG repeat RNA can cause deleterious effects in mammals. They also suggest the possible involvement of an RNA mechanism in human diseases with long CAG repeats.

  6. Effects of novelty stress on hippocampal gene expression, corticosterone and motor activity in mice.

    Kurumaji, Akeo; Umino, Masakazu; Nishikawa, Toru


    Exposure to novelty, a mild psychological stressor, induces neuronal activations in the hippocampus of rodents, which may play an important role in the adaptation to stress. We examined the changes in three parameters, i.e., gene expression in the hippocampus using a RT-PCR method, corticosterone and motor activity, in mice exposed to a new environment for 120min. A sharp and short-lasting increase in the gene expression of a set of stress-related genes previously reported, e.g., Fos and Nr4a1, was observed during the stress, with a similar pattern of changes in corticosterone. The motor activity gradually decreased during the novelty stress, indicating a process of adaptation to the new environment. In addition, in order to minimize the effects of elevated adrenal hormones by the stress, we carried out experiments on adrenalectomized (ADX) mice. However, the adrenalectomy produced minimal changes in the pattern and the magnitude of the gene response after the stress, while the motor activity showed a relatively slower pattern of adaptation in the ADX mice. Hence, the present study suggests that there was a coordinated adaptation process to the new environment in mice, and that the transcriptional response was mediated by neuronal networks rather than by adrenal hormones.

  7. Hepatocyte-specific IKKβ expression aggravates atherosclerosis development in APOE*3-Leiden mice

    Wong, Man C.; van Diepen, Janna A.; Hu, Lihui; Guigas, Bruno; de Boer, Hetty C.; van Puijvelde, Gijs H.; Kuiper, Johan; van Zonneveld, Anton J.; Shoelson, Steven E.; Voshol, Peter J.; Romijn, Johannes A.; Havekes, Louis M.; Tamsma, Jouke T.; Rensen, Patrick C.N.; Hiemstra, Pieter S.; Berbée, Jimmy F.P.


    Objective The liver is the key organ involved in systemic inflammation, but the relation between hepatic inflammation and atherogenesis is poorly understood. Since nuclear factor-κB (NF-κB) is a central regulator of inflammatory processes, we hypothesized that chronically enhanced hepatic NF-κB activation, through hepatocyte-specific expression of IκB kinase-β (IKKβ) (LIKK), will aggravate atherosclerosis development in APOE*3-Leiden (E3L) mice. Methods and Results E3L.LIKK and E3L control littermates were fed a Western-type diet for 24 weeks. E3L.LIKK mice showed a 2.3-fold increased atherosclerotic lesion area and more advanced atherosclerosis in the aortic root with less segments without atherosclerotic lesions (11 vs. 42%), and more segments with mild (63% vs. 44%) and severe (26% vs. 14 %) lesions. Expression of LIKK did not affect basal levels of inflammatory parameters, but plasma cytokine levels tended to be higher in E3L.LIKK mice after lipopolysaccharide (LPS) administration. E3L.LIKK mice showed transiently increased plasma cholesterol levels, confined to (V)LDL. This transient character resulted in a mild (+17%) increased cumulative plasma cholesterol exposure. Conclusion We conclude that selective activation of NF-κB in hepatocytes considerably promotes atherosclerosis development which is (at least partly) explained by an increased sensitivity to proinflammatory triggers and transiently increased plasma cholesterol levels. PMID:21798539

  8. Exercise Training Effects on Inflammatory Gene Expression in White Adipose Tissue of Young Mice

    Tracy Baynard


    Full Text Available We aimed to determine the effects of 6 wks of exercise on inflammatory markers in mice concomitantly fed either high-fat (HF or normal chow (NC diets in young mice. C57BL/6 mice were randomized into (/group an NC/sedentary (NC/SED, NC/exercise (NC/EX, HF/SED, and HF/EX groups. Treadmill exercise was performed 5 d/wk at 12 m/min, with 12% grade for 40 min/d. Liver triglycerides and gene expression of F4/80, MCP-1, TNF-α, leptin, and VEGF in visceral white adipose were determined. NC groups had lower body weights after 6 wks versus the HF groups ( versus  g (. F4/80 gene expression (indicator of macrophage infiltration and liver triglycerides were greatest amongst the HF/SED group, with no differences between the remaining groups. VEGF (indicator of angiogenesis was greatest in the HF/EX versus the other 3 groups (. Exposure of an HF diet in sedentary young mice increased visceral adipose depots and liver triglycerides versus an NC diet. Exercise training while on the HF diet protected against hepatic steatosis and possibly macrophage infiltration within white adipose tissue. This suggests that moderate exercise while on an HF diet can offer some level of protection early on in the development of obesity.

  9. Global renal gene expression profiling analysis in B2-kinin receptor null mice: impact of diabetes.

    Miran A Jaffa

    Full Text Available Diabetic nephropathy (DN, the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The risk factors and mechanisms that contribute to the development and progression of DN are still incompletely defined. To address the involvement of bradykinin B(2-receptors (B(2R in DN, we used a genome wide approach to study the effects of diabetes on differential renal gene expression profile in wild type and B(2R knockout (B(2R(-/- mice. Diabetes was induced with streptozotocin and plasma glucose levels and albumin excretion rate (AER were measured at predetermined times throughout the 23 week study period. Longitudinal analysis of AER indicated that diabetic B(2R(-/-D null mice had a significantly decreased AER levels compared to wild type B(2R(+/+D mice (P = 0.0005. Results from the global microarray study comparing gene expression profiles among four groups of mice respectively: (B(2R(+/+C, B(2R(+/+D, B(2R(-/-C and B(2R(-/-D highlighted the role of several altered pathological pathways in response to disruption of B(2R and to the diabetic state that included: endothelial injury, oxidative stress, insulin and lipid metabolism and inflammatory process with a marked alteration in the pro-apoptotic genes. The findings of the present study provide a global genomics view of biomarkers that highlight the mechanisms and putative pathways involved in DN.

  10. Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice.

    DeMayo, F J; Finegold, M J; Hansen, T N; Stanley, L A; Smith, B; Bullock, D W


    The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.

  11. Expression of the SET protein in testes of mice at different developmental stages

    Xiao-Nan Dai; Shan Liu; Li Shao; Chao Gao; Li Gao; Jia-Yin Liu; Yu-Gui Cui


    SET is a multifunctional protein involved in regulating many biological processes of the cell cycle. It is also a regulator of steroidogenesis in the ovary. However, the expression of SET protein in testis, and its function, still remains ambiguous. In this study, we observed the expression of SET in the testes of mice at different developmental stages, and have discussed its potential function in regulating spermatogenesis and androgen production. Forty‑eight male mice at different developmental stages(1week old as the infancy group; 4weeks old as the prepubertal group; 12weeks old as the adult group; over12months old as the ageing group) were used. Cellular location of SET protein in the testes was observed by immuno‑histochemistry. Expression levels of Set mRNA and SET protein were analyzed by quantitative polymerase chain reaction and Western blotting. SET protein was expressed in spermatogonial cells and spermatocytes; the highest level was mainly in haploid and tetraploid cells of the prepubertal and adult groups, and Leydig cells of the adult and ageing groups. There was a low expression in Sertoli cells. Expression of Set mRNA in the prepubertal group was signiifcantly higher than that in the adult group(P<0.05), while expression of SET protein was at the highest level in the adult group(P<0.05).SET protein is mainly expressed in spermatogonial cells and spermatocytes, and poorly expressed in Sertoli cells, suggesting that it is involved in spermatogenesis. Expression of SET protein in Leydig cells suggests a possible role in steroidogenesis.


    Charitidi, K.; Frisina, R. D.; Vasilyeva, O. N.; Zhu, X.; Canlon, B.


    Estrogens are important in the development, maintenance and physiology of the CNS. Several studies have shown their effects on the processing of hearing in both males and females, and these effects, in part, are thought to result from regulation of the transcription of genes via their classical estrogen receptor (ER) pathway. In order to understand the spatiotemporal changes that occur with age, we have studied the expression of ERs in the central auditory pathway in prepubertal and aged CBA mice with immunohistochemistry. In prepubertal mice a clear dichotomy was noted between the expression of ERα and ERβ. ERβ-positive neurons were found in the metencephalon whereas the majority of ERα was found in mesencephalon, diencephalon or the telencephalon. In the aged animals a different pattern of ER expression was found in terms of location and overall intensity. These age-induced changes in the expression pattern were generally not uniform, suggesting that region-specific mechanisms regulate the ERs’ age-related expression. Neither the prepubertal nor the aged animals showed sex differences in any auditory structure. Our results demonstrate different age-dependent spatial and temporal changes in the pattern of expression of ERα and ERβ, suggesting that each ER type may be involved in distinct roles across the central auditory pathway in different periods of maturation. PMID:20736049

  13. Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice

    Postina Rolf


    Full Text Available Abstract Background In a transgenic mouse model of Alzheimer disease (AD, cleavage of the amyloid precursor protein (APP by the α-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. Results To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the α-secretase ADAM10, or a dominant-negative mutant (dn of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant. Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice. Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients. Conclusion In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of

  14. Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

    Jianke Ren; Long Wang; Guoxiang Liu; Wen Zhang; Zhejin Sheng; Zhugang Wang; Jian Fei


    Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibodypreparation.

  15. Expression of Attractin in male reproductive tract of human and mice and its correlation with male reproduction.

    Cheng, Dan; Ming, Yu; Li, Jie; Chi, Yan; Li, Hong-Gang; Zou, Yu-Jie; Xiong, Cheng-Liang


    The expression of Attractin mRNA and protein in testis and semen of human and male mice was investigated. Human testis and semen samples were all collected from Reproductive Center of Renmin Hospital, Wuhan University in December, 2012. Testis samples were collected from 7 cases of obstructive azoospermias when they were subjected to diagnosed testis biopsy, and 30 normal human semen samples were obtained from those cases of semen analysis. Adult mice testis tissues were obtained from 10 2-month-old male BALB/c mice, and 60 male mice at different ages were classified into 10 groups (day 1, 5, 10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6 each). The expression of Attractin mRNA and protein in testis was detected by RT-PCR and Western blotting respectively. Human semen samples were centrifuged into sperm plasma (SP) and sperm extract (SE), and mice sperm samples were collected from the epididymis of 10 adult male BALB/c mice. Western blotting was used to determine the Attractin protein expression level. Attractin mRNA and protein were expressed in the testis of both patients with obstructive azoospermias and adult Bcl/B mice. Quantitative RT-PCR revealed that no Attractin mRNA was detectable in day 1 male BALB/c mice group. The Attractin mRNA and protein levels were low on the day 10, and increased with age until day 56. On the day 120, the expression levels of Attractin were decreased. As for human semen samples, Attractin protein was expressed in both SP and SE, but didn't exist in samples from the epididymis of male BALB/c mice. It was suggested that Attractin acted as a novel active substance and was involved in male reproduction in both human and BALB/c mice, but it exerted a different expression profile in different mammal species.

  16. In vivo imaging of Lactococcus lactis, Lactobacillus plantarum and Escherichiacoli expressing infrared fluorescent protein in mice

    Berlec, Aleš; Štrukelj, Borut; Završnik, Janja; Turk, Boris; Butinar, Miha


    Background In vivo imaging of orally administered lactic acid bacteria (LAB) and commensal bacteria in mice is shown to provide information on the spatial and temporal distribution of bacteria in the gastrointestinal tract. The bacteria can be detected and monitored using bioluminescence or near-infrared fluorescence. Results Fluorescence imaging of bacteria was established by expressing the infrared fluorescent protein IRFP713 in Lactococcus lactis, Lactobacillus plantarum and Escherichia co...

  17. Diagnosis of partial body radiation exposure in mice using peripheral blood gene expression profiles.

    Sarah K Meadows

    Full Text Available In the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI. However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79-100% compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16-43%, suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation.

  18. Nesfatin-1 stimulates cholecystokinin and suppresses peptide YY expression and secretion in mice.

    Ramesh, Naresh; Mortazavi, Sima; Unniappan, Suraj


    Nesfatin-1 is an 82 amino acid secreted peptide encoded in the precursor, nucleobindin-2 (NUCB2). It is an insulinotropic anorexigen abundantly expressed in the stomach and hypothalamus. Post-prandial insulin secretion is predominantly regulated by incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Nesfatin-1 was previously reported to modulate GLP-1 and GIP secretion in vitro in an enteroendocrine (STC-1) cell line. Intestine is a source of additional hormones including cholecystokinin (CCK) and peptide YY (PYY) that regulate metabolism. We hypothesized that nesfatin-1 modulates CCK and PYY secretion. Immunofluorescence histochemistry showed NUCB2/nesfatin-1 co-localizing CCK and PYY in the intestinal mucosa of mice. Static incubation of STC-1 cells with nesfatin-1 upregulated both CCK mRNA expression (1 and 10 nM) and secretion (0.1, 1 and 10 nM) at 1 h post-incubation. In contrast, nesfatin-1 treatment for 1 h downregulated PYY mRNA expression (all doses tested) and secretion (0.01 and 0.1 nM) in STC-1 cells. Continuous infusion of nesfatin-1 using osmotic mini-pumps for 12 h upregulated CCK mRNA expression in large intestine, and downregulated PYY mRNA expression in both large and small intestines of male C57BL/6J mice. In these tissues, Western blot analysis found a corresponding increase in CCK and a decrease in PYY content. Collectively, we provide new information on the cell specific localization of NUCB2/nesfatin-1 in the intestinal mucosa, and a novel function for nesfatin-1 in modulating intestinal CCK and PYY expression and secretion in mice.

  19. Expression of Galectin-3 As A Testis Inflammatory Marker in Vasectomised Mice

    Homayoun Naderian


    Full Text Available Objective: Vasectomy, though in some cases are being confronted with irreversibility, has been accepted as an effective contraceptive method. It is estimated that near 2-6% of vasectomised men ultimately show a tendency to restore their fertility. In some cases, vasectomy has been considered as an irreversible procedure due to many post-vasectomy complications causing this debate. The aim of present study was to investigate the pattern of expression of galectin-3, an inflammatory factor secreted by macrophages and immune cells, following the vasectomy in mice testis tissue.Materials and Methods: In this experimental study, twenty mature male Balb/c mice, aged two months, were divided into two equal groups: sham and vasectomised groups (n=10. They were sacrificed four months after vasectomy, while the pattern of galectin-3 expression was investigated using a standard immunohistochemistry technique on testicular tissues. Stereological analyses of testes parameters in vasectomised and sham-operated groups were compared by mixed model analysis.Results: Based on observations, although galectin-3 was not expressed in sham-operated group, it was expressed in 40% of testicular tissues of vasectomised mice, like: seminiferous tubules, interstitial tissues and tunica albugina. Also, our result showed a significant alteration in number of germ and sertoli cells of testicular tissue in vasectomised group in comparison to sham-operated group. In addition, the result of mixed model method confirmed a significant reduction in germ and sertoli cells of vasectomised group.Conclusion: The expression of galectin-3 at different parts of testicular tissue in vasectomised group is higher than sham group. This express illustrates the increase of degenerative changes and inflammation reactions in testicular tissue, leading to chronic complications and infertility, after the vasovasostomy.

  20. Expression of Robo2 gene during the renal development in mice

    Jia-yao JI


    Full Text Available Objective To observe the expression of Robo2 gene, and explore its role during the renal development of mice. Methods Real-time quantitative RT-PCR was used to semi-quantitatively measure the expression level of Robo2 mRNA in the developing murine kidney at fetal age of 12.5, 13.5, 14.5, 15.5, 16.5 and 17.5 days, and also 1 day, 1 week, 5 weeks after birth. Immunofluorescence staining was used to examine the expression location of Robo2 protein at different stages of embryonic and postnatal kidney. Results Real-time quantitative RT-PCR analysis revealed that Robo2 was highly expressed in embryonic kidney at fetal age of 12.5, 13.5 and 14.5 days, while the expression level declined quickly thereafter and maintained at very low level after birth. Immunofluorescence staining showed that the expression of Robo2 protein could be primarily detected in metanephric mesenchyme of the developing kidney, but not in the ureteric bud. With the development of embryonic kidney, Robo2 protein was expressed in cell membrane of metanephric mesenchyme, condensed cap mesenchyme surrounding the tip of the ureteric bud, comma-shaped body, S-shaped body and renal capsule, finally expressed in the podocytes. Besides, Robo2 protein was also weakly expressed in part of the proximal tubular epithelial cells. Absence of Robo2 gene resulted in abnormal development of nephron, and broadening of some renal tubules and collecting ducts. Conclusion Robo2 plays an important role in the nephron development in mice by regulating the interaction of metanephric mesenchyme and ureteric bud.

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

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


    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.

  2. Expression of Interleukin-17A in Lung Tissues of Irradiated Mice and the Influence of Dexamethasone

    Li-Ping Wang


    Full Text Available Purpose. To investigate the expressions of IL-17A in different phases of radiation-induced lung injury and the effect of dexamethasone. Methods. The thorax of C57BL/6 mice was irradiated with 15 Gy rays. Mice from dexamethasone-treated group were injected intraperitoneally with dexamethasone (0.42 mg/kg/day every day for the first month after irradiation. IL-17A in lung tissues was detected by immunohistochemistry. IL-17A, TGF-β1, and IL-6 in bronchoalveolar lavage fluid were detected by ELISA. Lung inflammation and collagen deposition were observed by H&E and Masson methods. The degree of alveolitis and fibrosis was judged according to scoring. Results. IL-17A expression was appreciable at 1 week, peaked at 4 weeks, and subsequently declined at 8 weeks after irradiation. IL-17A was reduced after dexamethasone application at all the observation periods. Dexamethasone also inhibited expressions of TGF-β, IL-6, and TNF-α in bronchoalveolar lavage fluid. Moreover, dexamethasone attenuated the severity of lung injury by reducing the infiltration of inflammatory cells and collagen deposition. Terms of survival and the time of death in mice of treatment group were postponed and survival rate was improved. Conclusions. IL-17A plays an important role in the process of radiation-induced lung injury. And dexamethasone may provide a protective role in lung injury induced by radiation.

  3. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Li, Dejia; Lai, Yi; Yue, Yongping; Rabinovitch, Peter S; Hakim, Chady; Duan, Dongsheng


    Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT) was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12) vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy) and ameliorate muscle disease.

  4. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Dejia Li

    Full Text Available Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12 vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy and ameliorate muscle disease.

  5. BALB/c mice deficient in CD4 T cell IL-4Rα expression control Leishmania mexicana Load although female but not male mice develop a healer phenotype.

    Karen J Bryson


    Full Text Available Immunologically intact BALB/c mice infected with Leishmania mexicana develop non-healing progressively growing lesions associated with a biased Th2 response while similarly infected IL-4Rα-deficient mice fail to develop lesions and develop a robust Th1 response. In order to determine the functional target(s for IL-4/IL-13 inducing non-healing disease, the course of L. mexicana infection was monitored in mice lacking IL-4Rα expression in specific cellular compartments. A deficiency of IL-4Rα expression on macrophages/neutrophils (in LysM(creIL-4Rα(-/lox animals had minimal effect on the outcome of L. mexicana infection compared with control (IL-4Rα(-/flox mice. In contrast, CD4(+ T cell specific (Lck(creIL-4Rα(-/lox IL-4Rα(-/- mice infected with L. mexicana developed small lesions, which subsequently healed in female mice, but persisted in adult male mice. While a strong Th1 response was manifest in both male and female CD4(+ T cell specific IL-4Rα(-/- mice infected with L. mexicana, induction of IL-4 was manifest in males but not females, independently of CD4(+ T cell IL-4 responsiveness. Similar results were obtained using pan-T cell specific (iLck(creIL-4Rα(-/lox IL-4Rα(-/- mice. Collectively these data demonstrate that upon infection with L. mexicana, initial lesion growth in BALB/c mice is dependent on non-T cell population(s responsive to IL-4/IL-13 while progressive infection is dependent on CD4(+ T cells responsive to IL-4.

  6. Expression of mdr isoforms in mice during estrous cycle and under hormone stimulation

    Marion Schiengold


    Full Text Available The multidrug resistance (MDR phenotype is associated with the expression of P-glycoprotein (Pgp, coded by the multigenic mdr family. Mice present the isoforms mdr1 and mdr3, which are responsible for multidrug resistance, and mdr2, that is involved in the transport of phospholipids. mdr1 expression has more recently been associated also with the secretion of steroid hormones. This work presents an RT-PCR analysis of the expression of mdr isoforms, in several organs of mice during different phases of the estrous cycle. Additionally, females were ovariectomized, submitted to different hormone treatments, and their uterus was analyzed for the expression of mdr isoforms. The results show that in the adrenal gland and ovaries mdr1 is the main isoform during proestrus, and that progesterone or a combination of progesterone and estrogen induce the expression of all mdr isoforms in the uterus of ovariectomized females. We suggest that the functions of mdr1 and mdr3 are overlapping, that mdr3 may be the more efficient isoform in the detoxification function, and that mdr1 may be more closely related to the secretion of steroid hormones.

  7. Effects of Ligustrazine on Expression of Bone Marrow Heparan Sulfates in Syngeneic Bone Marrow Transplantation Mice

    任天华; 刘文励; 孙汉英; 戴琪琳; 孙岚


    To explore the effects of ligustrazine on bone marrow heparan sulfates (HS) expression in bone marrow transplantation (BMT) mice, the syngeneic BMT mice were orally given 2 mg ligustrazine twice a day. On the 7th, 10th, 14th, 18th day after BMT, peripheral blood cells and bone marrow nuclear cells (BMNC) were counted, and the expression levels of HS in bone marrow and on the stromal cell surfaces were detected by immunohistochemistry and flow cytometry assay respectively. In ligustrazine-treated group, the white blood cells (WBC) and BMNC on the 7th, 10th, 14th, 18th day and platelets (PLT) on the 7th, 10th day were all significantly more than those in control group (P<0.05). The bone marrow HS expression levels in ligustrazine-treated group were higher than those in control group (P<0. 05) on the 7th, 10th, 14th, 18th day. However, the HS expression levels on the stromal cell surfaces showed no significant difference between the two groups on the 18th day (P>0. 05). It was concluded that ligustrazine could up-regulate HS expression in bone marrow, which might be one of the mechanisms contributing to ligustrazine promoting hematopoietic reconstitution after BMT.

  8. Gene expression analysis of pancreatic cystic neoplasm in SV40Tag transgenic mice model

    Jie Feng; Qiang Sun; Cheng Gao; Juan Dong; Xiao-Luan Wei; Hua Xing; Hou-Da Li


    AIM: To study the gene expression changes in pancreatic cystic neoplasm in SV40Tag transgenic mice model and to provide information about the prevention,clinical diagnosis and therapy of pancreatic cancer.METHODS: Using the pBC-SV40Tag transgenic mice model of pancreatic cystic neoplasm, we studied the gene expression changes by applying high-density microarrays. Validation of part gene expression profiling data was performed using real-time PCR.RESULTS: By using high-density oligonucleotide microarray, of 14113 genes, 453 were increased and 760 decreased in pancreatic cystic neoplasm, including oncogenes, cell-cycle-related genes, signal transduction-related genes, skeleton-related genes and metabolism-related genes. Among these, we confirmed the changes in Igf, Shh and Wnt signal pathways with real-time PCR.The results of real-time PCR showed similar expression changes in gene chip.CONCLUSION: all the altered expression genes are associated with cell cycle, DNA damage and repair, signal pathway, and metabolism. SV40Tag may cooperate with several proteins in promoting tumorigenesis.

  9. Dietary Nitrate Is a Modifier of Vascular Gene Expression in Old Male Mice

    Christos Rammos


    Full Text Available Aging leads to a number of disadvantageous changes in the cardiovascular system. Deterioration of vascular homoeostasis with increase in oxidative stress, chronic low-grade inflammation, and impaired nitric oxide bioavailability results in endothelial dysfunction, increased vascular stiffness, and compromised arterial-ventricular interactions. A chronic dietary supplementation with the micronutrient nitrate has been demonstrated to improve vascular function. Healthy dietary patterns may regulate gene expression profiles. However, the mechanisms are incompletely understood. The changes that occur at the gene expression level and transcriptional profile following a nutritional modification with nitrate have not been elucidated. To determine the changes of the vascular transcriptome, we conducted gene expression microarray experiments on aortas of old mice, which were treated with dietary nitrate. Our results highlight differentially expressed genes overrepresented in gene ontology categories. Molecular interaction and reaction pathways involved in the calcium-signaling pathway and the detoxification system were identified. Our results provide novel insight to an altered gene-expression profile in old mice following nitrate supplementation. This supports the general notion of nutritional approaches to modulate age-related changes of vascular functions and its detrimental consequences.

  10. IFN-{gamma} gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O. [Univ. of Alberta, Edmonton (Canada)] [and others


    Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet {beta}-cells following islet filtration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in {beta}-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1{beta}, IL-2, IL-4, IL-10, and IFN-{gamma} in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (>13 wk). However, only IFN-{gamma} mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-{gamma} and IL-4 were not different in the four groups of mice. These results suggest that islet {beta}-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-{gamma} production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-{gamma} production in the islets. 56 refs., 4 figs., 3 tabs.

  11. Isoforms of the nuclear envelope protein Nurim are differentially expressed during heart development in mice.

    Zhang, Wan; Bai, Tianyu; Zhang, Shuai; Xu, Shiqiang; Chen, Hengling; Li, Chenhong


    To date, transcript variants of the nuclear envelope protein Nurim and their expression profiles in mice have never been elucidated. In this study, we determined that the primary Nurim variant a was abundantly expressed in mouse heart, liver, spleen and kidney. The protein level of isoform a is initiated at an early stage of heart formation and demonstrated a significant increase in expression throughout embryonic heart development. Interestingly, Nurim b is also up-regulated from E12.5 to E18.5 in different individuals. Our research represents the first report on alternative splicing variants of mouse Nurim and their differential expression profile during embryonic development. These studies suggest a potential role for Nurim in early heart morphogenesis and should help further elucidate the function of Nurim. Copyright © 2017. Published by Elsevier B.V.

  12. Pilocarpine-induced status epilepticus alters hippocampal PKC expression in mice.

    Liu, Jian Xin; Liu, Yong; Tang, Feng Ru


    We investigated the protein expression of different protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta1, PKC-beta2, PKC-gamma, PKC-delta, PKC-epsilon, PKC-eta and PKC-zeta) in the hippocampus of normal control mice and progressive changes in PKC isoforms expression during and after pilocarpine induced status epilepticus (PISE). We showed the reduced expression of PKC-delta, PKC-eta and PKC-zeta in interneurons in the CA1 area and in the hilus of the dentate gyrus during or after PISE. Increased expression of PKC-alpha and PKC-beta1 was demonstrated in the stratum pyramidale of CA3 area, and PKC-epsilon was up-regulated in the stratum lucidum of the CA3 area during or after PISE. Our results suggest that hippocampal PKC isoforms may play different roles in seizure generation, and be targets for development of anti-convulsive drugs.

  13. The influence of bovine milk high or low in isoflavones on hepatic gene expression in mice

    Skaanild, Mette Tingleff; Nielsen, Tina Skau


    in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17β-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array q......Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes......PCR kit. It was revealed that Tween 80 and 17β-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones...

  14. A chronic high-cholesterol diet paradoxically suppresses hepatic CYP7A1 expression in FVB/NJ mice[S

    Henkel, Anne S.; Anderson, Kristy A.; Dewey, Amanda M.; Kavesh, Mark H.; Green, Richard M.


    Cholesterol 7α-hydroxylase (CYP7A1) encodes for the rate-limiting step in the conversion of cholesterol to bile acids in the liver. In response to acute cholesterol feeding, mice upregulate CYP7A1 via stimulation of the liver X receptor (LXR) α. However, the effect of a chronic high-cholesterol diet on hepatic CYP7A1 expression in mice is unknown. We demonstrate that chronic cholesterol feeding (0.2% or 1.25% w/w cholesterol for 12 weeks) in FVB/NJ mice results in a >60% suppression of hepati...

  15. 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.

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K; Meuleman, Philip; Serre, Stephanie B N; Lademann, Jacob B; Ghanem, Lubna; Scheel, Troels K H; Leroux-Roels, Geert; Bukh, Jens


    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) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log(10) FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.

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

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


    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.

  17. Stress induces altered CRE/CREB pathway activity and BDNF expression in the hippocampus of glucocorticoid receptor-impaired mice.

    Alboni, Silvia; Tascedda, Fabio; Corsini, Daniela; Benatti, Cristina; Caggia, Federica; Capone, Giacomo; Barden, Nicholas; Blom, Joan M C; Brunello, Nicoletta


    The gene coding for the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) is a stress-responsive gene. Changes in its expression may underlie some of the pathological effects of stress-related disorders like depression. Data on the stress-induced regulation of the expression of BDNF in pathological conditions are rare because often research is conducted using healthy animals. In our experiments, we used transgenic mice with glucocorticoid receptor impaired (GR-i) expression in the hypothalamus created as a tool to study the neuroendocrine changes occurring in stress-related disorders. First, under basal condition, GR-i mice displayed lower levels of BDNF exons IX and IV and decreased CRE(BDNF) binding activity with respect to wild-type (WT) mice in the hippocampus. Then, we exposed GR-i and WT mice to an acute restraint stress (ARS) to test the hypothesis that GR-i mice display: 1] different ARS induced expression of BDNF, and 2] altered activation of signaling pathways implicated in regulating BDNF gene expression in the hippocampus with respect to WT mice. Results indicate that ARS enhanced BDNF mRNA expression mainly in the CA3 hippocampal sub-region of GR-i mice in the presence of enhanced levels of pro-BDNF protein, while no effect was observed in WT mice. Moreover, ARS reduced CREB signaling and binding to the BDNF promoter in GR-i mice but enhanced signaling and binding, possibly through ERK1/2 activation, in WT mice. Thus, life-long central GR dysfunction resulted in an altered sensitivity at the transcriptional level that may underlie an impaired response to an acute psycho-physical stress. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

  18. Immuno-protection of SJIR-2 DNA vaccine with microspheres adjuvant in mice challenged with Schistosoma japonicum%日本血吸虫SJIR-2纳米微球核酸疫苗的免疫保护性研究

    王正印; 潘丽红; 汪学龙


    目的:研究日本血吸虫胰岛素受体-2( SJIR-2)纳米微球核酸疫苗对小鼠攻击感染的免疫保护效果。方法构建pEGFP-SJIR-2重组质粒,双酶切鉴定并测序,大量提取pEGFP-SJIR-2质粒,用壳聚糖( CHS)修饰的聚乳酸-羟基乙酸共聚物( PLGA)微球包裹,用包裹后的SJIR-2纳米微球免疫小鼠。将40只雌性BALB/c小鼠随机分为4组( n=10),分别注射 PBS、空 pEGFP 质粒、CHS-PLGA 微球和 CHS-PL-GA-pEGFP-SJIR-2微球各100μg免疫小鼠,末次免疫2周后,用日本血吸虫尾蚴攻击感染小鼠,每次免疫及感染尾蚴前收集各组小鼠血清,ELISA法检测各组小鼠血清内免疫球蛋白( IgG)水平的变化。小鼠感染尾蚴42 d后全部剖杀,收集成虫和虫卵并计算减虫率和减卵率。结果成功构建了pEGFP-SJIR-2重组质粒,与PBS组比较, CHS-PLGA-pEGFP-SJIR-2组的成虫数和虫卵数差异有统计学意义( P<0.01)。CHS-PLGA-pEGFP-SJIR-2组的减虫率和减卵率分别为37.36%和46.82%,和PBS组相比,CHS-PLGA-pEGFP-SJIR-2组小鼠血清内IgG水平比明显增高( P<0.01),而pEGFP组和CHS-PLGA组成虫数和虫卵数与PBS组比较差异无统计学意义。结论 SJIR-2纳米微球核酸疫苗对感染血吸虫的BALB/c小鼠有一定的免疫保护效果,对其潜在的候选抗原疫苗的价值尚需深入研究。%Objective To research the immuno-protection of SJIR-2 DNA vaccine with nanometer microspheres a-gainst Schistosoma japonicum infection in mice. Methods To construct eukaryotic expression plasmid pEGFP-SJIR-2, identified by double digestion and sequenced delivery. The recombinant plasmid pEGFP-SJIR-2 was ex-tracted and was encapsulated into PLGA nanometer microspheres which were modified by CHS. 40 female BALB/c mice were randomly divided into 4 groups (n=10), each group of mice were injected with PBS, empty pEGFP plasmid, CHS-PLGA nanometer microspheres and CHS-PLGA-pEGFP-SJIR-2 nanometer microspheres 100 μg, re-spectively. Two weeks

  19. CRF receptor antagonist astressin-B reverses and prevents alopecia in CRF over-expressing mice.

    Lixin Wang

    Full Text Available Corticotropin-releasing factor (CRF signaling pathways are involved in the stress response, and there is growing evidence supporting hair growth inhibition of murine hair follicle in vivo upon stress exposure. We investigated whether the blockade of CRF receptors influences the development of hair loss in CRF over-expressing (OE-mice that display phenotypes of Cushing's syndrome and chronic stress, including alopecia. The non-selective CRF receptors antagonist, astressin-B (5 µg/mouse injected peripherally once a day for 5 days in 4-9 months old CRF-OE alopecic mice induced pigmentation and hair re-growth that was largely retained for over 4 months. In young CRF-OE mice, astressin-B prevented the development of alopecia that occurred in saline-treated mice. Histological examination indicated that alopecic CRF-OE mice had hair follicle atrophy and that astressin-B revived the hair follicle from the telogen to anagen phase. However, astressin-B did not show any effect on the elevated plasma corticosterone levels and the increased weights of adrenal glands and visceral fat in CRF-OE mice. The selective CRF₂ receptor antagonist, astressin₂-B had moderate effect on pigmentation, but not on hair re-growth. The commercial drug for alopecia, minoxidil only showed partial effect on hair re-growth. These data support the existence of a key molecular switching mechanism triggered by blocking peripheral CRF receptors with an antagonist to reset hair growth in a mouse model of alopecia associated with chronic stress.

  20. Expression of adipose microRNAs is sensitive to dietary conjugated linoleic acid treatment in mice.

    Pilar Parra

    Full Text Available BACKGROUND: Investigation of microRNAs (miRNAs in obesity, their genetic targets and influence by dietary modulators is of great interest because it may potentially identify novel pathways involved in this complex metabolic disorder and influence future therapeutic approaches. This study aimed to determine whether miRNAs expression may be influenced by conjugated linoleic acid (CLA, currently used to induce fat loss. METHODOLOGY/PRINCIPAL FINDINGS: We determined retroperitoneal adipose tissue (rWAT expression of five miRNAs related to adipocyte differentiation (miRNA-143 and lipid metabolism (miRNA-103 and -107 and altered in obesity (miRNA-221 and -222, using the TaqMan®MicroRNA Assay (Applied-Biosystems. In the first experiment, mice were fed with a standard fat diet and orally treated with sunflower oil (control group and 3 or 10 mg CLA/day for 37 days. In the second experiment, mice were fed with a high fat diet for 65 days. For the first 30 days, mice received the same doses of CLA described above and, from that time onwards, animals received a double dose. Results showed that expression of selected miRNAs was modified in response to CLA treatment and metabolic status. Interestingly, a strong correlation was observed between miR-103 and -107 expression, as well as miR-221 and -222 in both experiments. Moreover, changes in miRNAs expression correlated with several adipocyte gene expressions: miR-103 and -107 correlated with genes involved in fatty acid metabolism whereas miR-221 and miR-222 correlated with the expression of adipocytokines. Regarding the minor changes observed in miR-143 expression, no differences in expression of adipogenic markers were observed. CONCLUSIONS/SIGNIFICANCE: Although elucidating the functional implications of miRNAs is beyond the scope of this study, these findings provide the first evidence that miRNAs expression may be influenced by dietary manipulation, reflecting or even contributing to the new metabolic

  1. Effect of ECR on the Expression of Tau from the Brain of the Mice Induced by Overload Aluminum Salt

    YANGSu-Fen; WUZhong-Jun; YANGZheng-Qin; LIYu; WuQin; ZHOUQi-Xin; SHIJing-Shan


    Aim: To study the effect of Ecdysterone (ECR) on the expression of Tau from the cerebral cortice and hippocampus and behaviors in passive avoidance reaction and spatial discrimination of the mice induced by overload aluminum salt.Methods Fourty-five NIH mice were randomly divided into five groups, the control group, the model group, the treated

  2. Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

    卢一凡; 田靫; 邓继先; 程萱; 黄培堂


    Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du

  3. Hierarchy in the home cage affects behaviour and gene expression in group-housed C57BL/6 male mice.

    Horii, Yasuyuki; Nagasawa, Tatsuhiro; Sakakibara, Hiroyuki; Takahashi, Aki; Tanave, Akira; Matsumoto, Yuki; Nagayama, Hiromichi; Yoshimi, Kazuto; Yasuda, Michiko T; Shimoi, Kayoko; Koide, Tsuyoshi


    Group-housed male mice exhibit aggressive behaviour towards their cage mates and form a social hierarchy. Here, we describe how social hierarchy in standard group-housed conditions affects behaviour and gene expression in male mice. Four male C57BL/6 mice were kept in each cage used in the study, and the social hierarchy was determined from observation of video recordings of aggressive behaviour. After formation of a social hierarchy, the behaviour and hippocampal gene expression were analysed in the mice. Higher anxiety- and depression-like behaviours and elevated gene expression of hypothalamic corticotropin-releasing hormone and hippocampal serotonin receptor subtypes were observed in subordinate mice compared with those of dominant mice. These differences were alleviated by orally administering fluoxetine, which is an antidepressant of the selective serotonin reuptake inhibitor class. We concluded that hierarchy in the home cage affects behaviour and gene expression in male mice, resulting in anxiety- and depression-like behaviours being regulated differently in dominant and subordinate mice.

  4. Ovariectomy upregulated the expression of Peroxiredoxin 1 & 5 in osteoblasts of mice

    Du, Juan; Feng, Wei; Sun, Jing; Kang, Cuijie; Amizuka, Norio; Li, Minqi


    Peroxiredoxin (PRX), a family of peroxidases, is associated with various biological processes such as the detoxification of oxidants and cell apoptosis. Besides, the anti-apoptosis effect of estrogen results partially from its anti-oxidant function. The purpose of this study was to investigate the expression of PRXs in ovariectomy (OVX) mice and the related anti-oxidative mechanism of estrogen. Eight-week-old mice were subjected to ovariectomy. MC3T3-E1 cells were pretreatment with 17b-estradiol and N-acetyl cysteine followed by oxidative injury induced with H2O2. Western blot and real time-PCR were applied to clarify the expressions of PRX1 and caspase-3, with both wild-type and PRX1 knockout MC3T3-E1 cells generated by CRISPR/Cas9 technology. The results showed PRX1 and PRX5 were upregulated in osteoblasts in the proximal tibial metaphysis of ovariectomy mice. Interestingly, PRX1 and PRX5 showed different distribution patterns, with PRX1 mainly accumulated in cell nuclei and PRX5 in the cytoplasm. Gene expression analysis showed significantly reduced expressions of PRX1 and caspase-3 in the pretreatment groups when compared with cells treated with H2O2 alone. Also, a decrease of caspase-3 expressions was observed in PRX1 knockout MC3T3-E1 cells with or without H2O2 in comparison to wild-type cells. These findings suggested that PRX may play important roles in estrogen-deficient osteoporosis. (200 words). PMID:27786251

  5. Changes in photoperiod alter Glut4 expression in skeletal muscle of C57BL/6J mice.

    Tashiro, Ayako; Shibata, Satomi; Takai, Yusuke; Uchiwa, Tatsuhiro; Furuse, Mitsuhiro; Yasuo, Shinobu


    Seasonal changes in photoperiod influence body weight and metabolism in mice. Here, we examined the effect of changes in photoperiod on the expression of glucose transporter genes in the skeletal muscle and adipose tissue of C57BL/6J mice. Glut4 expression was lower in the gastrocnemius muscle of mice exposed to a short-duration day (SD) than those to a long-duration day (LD), with accompanying changes in GLUT4 protein levels. Although Glut4 expression in the mouse soleus muscle was higher under SD than under LD, GLUT4 protein levels remained unchanged. To confirm the functional significance of photoperiod-induced changes in Glut4 expression, we checked for variations in insulin sensitivity. Blood glucose levels after insulin injection remained high under SD, suggesting that the mice exposed to SD showed lower sensitivity to insulin than those exposed to LD. We also attempted to clarify the relationship between Glut4 expression and physical activity in the mice following changes in photoperiod. Locomotor activity, as detected via infrared beam sensor, was lower under SD than under LD. However, when we facilitated voluntary activity by using running wheels, the rotation of wheels was similar for both groups of mice. Although physical activity levels were enhanced due to running wheels, Glut4 expression in the gastrocnemius muscle remained unchanged. Thus, variations in photoperiod altered Glut4 expression in the mouse skeletal muscle, with subsequent changes in GLUT4 protein levels and insulin sensitivity; these effects might be independent of physical activity.

  6. Ginsenoside Rb1 ameliorates liver fat accumulation by upregulating perilipin expression in adipose tissue of db/db obese mice

    Xizhong Yu


    Conclusion: G-Rb1 may improve insulin sensitivity in obese and diabetic db/db mice by reducing hepatic fat accumulation and suppressing adipocyte lipolysis; these effects may be mediated via the upregulation of perilipin expression in adipocytes.

  7. Regulation of endogenous glucose production in glucose transporter 4 over-expressing mice.

    Eric D Berglund

    Full Text Available Strategies to amplify whole-body glucose disposal are key therapies to treat type 2 diabetes. Mice that over-express glucose transporter 4 (Glut4 in skeletal muscle, heart, and adipose tissue (G4Tg exhibit increased fasting glucose disposal and thus lowered blood glucose. Intriguingly, G4Tg mice also exhibit improved insulin-stimulated suppression of endogenous glucose production even though Glut4 is not present in the liver. It is unclear, however, if hepatic gluco-regulation is altered in G4Tg mice in the basal, non-insulin-stimulated state. The current studies were performed to examine fasting hepatic glucose metabolism in G4Tg mice and to determine whether gluco-regulatory adaptations exist in the non-insulin-stimulated condition. To test this question, phloridzin-glucose clamps were used to match blood glucose and pancreatic hormone levels while tracer dilution techniques were used to measure glucose flux. These techniques were performed in chronically-catheterized, conscious, and un-stressed 5h-fasted G4Tg and wild-type (WT littermates. Results show reduced blood glucose, hepatic glycogen content, and hepatic glucokinase (GK activity/expression as well as higher endogenous glucose production, glucose disposal, arterial glucagon, and hepatic glucose-6-phosphatase (G6Pase activity/expression in G4Tg mice versus WT controls. Clamping blood glucose for 90 min at ~115 mg/dLin G4Tg and WT mice normalized nearly all variables. Notably, however, net hepatic glycogen synthetic rates were disproportionately elevated compared to changes in blood glucose. In conclusion, these studies demonstrate that basal improvements in glucose tolerance due to increased uptake in extra-hepatic sites provoke important gluco-regulatory adaptations in the liver. Although changes in blood glucose underlie the majority of these adaptations, net hepatic glycogen synthesis is sensitized. These data emphasize that anti-diabetic therapies that target skeletal muscle, heart

  8. Transgenic mice expressing constitutive active MAPKAPK5 display gender-dependent differences in exploration and activity

    Moens Ugo


    Full Text Available Abstract Background The mitogen-activated protein kinases, MAPKs for short, constitute cascades of signalling pathways involved in the regulation of several cellular processes that include cell proliferation, differentiation and motility. They also intervene in neurological processes like fear conditioning and memory. Since little remains known about the MAPK-Activated Protein Kinase, MAPKAPK5, we constructed the first MAPKAPK knockin mouse model, using a constitutive active variant of MAPKAPK5 and analyzed the resulting mice for changes in anxiety-related behaviour. Methods We performed primary SHIRPA observations during background breeding into the C57BL/6 background and assessed the behaviour of the background-bred animals on the elevated plus maze and in the light-dark test. Our results were analyzed using Chi-square tests and homo- and heteroscedatic T-tests. Results Female transgenic mice displayed increased amounts of head dips and open arm time on the maze, compared to littermate controls. In addition, they also explored further into the open arm on the elevated plus maze and were less active in the closed arm compared to littermate controls. Male transgenic mice displayed no differences in anxiety, but their locomotor activity increased compared to non-transgenic littermates. Conclusion Our results revealed anxiety-related traits and locomotor differences between transgenic mice expressing constitutive active MAPKAPK5 and control littermates.

  9. Saccharomyces cerevisiae expressing Gp43 protects mice against Paracoccidioides brasiliensis infection.

    Mariana Aprigio Assis-Marques

    Full Text Available The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM. It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.

  10. Incubation and application of transgenic green fluorescent nude mice in visualization studies on glioma tissue remodeling

    DONG Jun; LAN Qing; HUANG Qiang; DAI Xing-liang; LU Zhao-hui; FEI Xi-feng; CHEN Hua; ZHANG Quan-bin; ZHAO Yao-dong; WANG Zhi-min; WANG Ai-dong


    Background The primary reasons for local recurrence and therapeutic failure in the treatment of malignant gliomas are the invasion and interactions of tumor cells with surrounding normal brain cells.However,these tumor cells are hard to be visualized directly in histopathological preparations,or in experimental glioma models.Therefore,we developed an experimental human dual-color in vivo glioma model,which made tracking solitary invasive glioma cells possible,for the purpose of visualizing the interactions between red fluorescence labeled human glioma cells and host brain cells.This may offer references for further studying the roles of tumor microenvironment during glioma tissue remodeling.Methods Transgenic female C57BL/6 mice expressing enhanced green fluorescent protein (EGFP) were crossed with male Balb/c nude mice.Then sib mating was allowed to occur continuously in order to establish an inbred nude mice strain with 50% of their offspring that are EGFP positive.Human glioma cell lines U87-MG and SU3 were transfected with red fluorescent protein (RFP) gene,and a rat C6 glioma cell line was stained directly with CM-Dil,to establish three glioma cell lines emitting red fluorescence (SU3-RFP,U87-RFP,and C6-CM-Dil).Red fluorescence tumor cells were inoculated via intra-cerebral injection into caudate nucleus of the EGFP nude mice.Tumor-bearing mice were sacrificed when their clinical symptoms appeared,and the whole brain was harvested and snap frozen for further analysis.Confocal laser scanning microscopy was performed to monitor the mutual interactions between tumor cells and host brain cells.Results Almost all the essential tissues of the established EGFP athymic Balb/c nude mice,except hair and erythrocytes,fluoresced green under excitation using a blue light-emitting flashlight with a central peak of 470 nm,approximately 50% of the offsprings were nu/nu EGFP+.SU3-RFP,U87-RFP,and C6-CM-Dil almost 100% expressed red fluorescence under the fluorescence

  11. Generation and characterization of transgenic mice expressing tamoxifen-inducible cre-fusion protein specifically in mouse liver.

    Zhu, Huan-Zhang; Chen, Jian-Quan; Cheng, Guo-Xiang; Xue, Jing-Lun


    To establish transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase specifically in the liver and to provide an efficient animal model for studying gene function in the liver and creating various mouse models mimicking human diseases. Alb-Cre-ERt transgenic mice were produced by microinjecting the construct with Cre-ERt fusion gene of DNA fragments into fertilized eggs derived from inbred C57BL/6 strain. Transgenic mice were identified by using PCR and Southern blotting. Expression of Cre-ERt fusion gene was analyzed in the liver, kidney, brain and lung from F1 generation transgenic mice at 8 weeks of age by reverse transcription (RT)-PCR. Four hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant Alb-Cre-ERt DNA fragments, and 312 survival eggs injected were transferred to the oviducts of 12 pseudopregnant recipient mice, 6 of 12 recipient mice became pregnant and gave birth to 44 offsprings. Of the 44 offsprings, two males and one female carried the hybrid Cre-ERt fusion gene. Three mice were determined as founders, and were back crossed to set up F1 generations with other inbred C57BL/6 mice. Transmission of Cre-ERt fusion gene in F1 offspring followed Mendelian rules. The expression of Cre-ERt mRNA was detected only in the liver of F1 offspring from two of three founder mice. Transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase under control of the liver-specific promoter are preliminary established.

  12. Expression of c-fos and c-jun protooncogenes in the uteri of immature mice neonatally exposed to diethylstilbestrol.

    Yamashita, S; Takayanagi, A; Shimizu, N


    We studied the cell-type-specific and temporal expression of c-fos and c-jun protooncogenes after 17beta-estradiol (E2) stimulation in the uteri of immature 3-week-old mice neonatally exposed to diethylstilbestrol (DES), DES-mice, and the ontogenic expression of these genes in the uteri of DES-mice using immunohistochemistry and in situ hybridization. A single E2 injection induced the transient and rapid expression of c-fos mRNA and c-Fos protein in the endometrial epithelium and endothelial cells of the blood vessels in both 3-week-old vehicle-treated controls and DES-mice; a peak of mRNA expression was 2 hours after E2 injection and that of protein expression was 2 to 3 hours after the injection. The expression of c-fos mRNA and protein after E2 stimulation was lower in the DES-mice than in the control animals. There were no significant differences in the c-jun expression patterns in both experimental groups before and after the E2 injection. The E2 injection transiently down-regulated the c-jun expression in the epithelium and up-regulated it in the stroma and myometrium. The uterine epithelium of DES-mice showed much stronger c-Jun immunostaining on days 4 and 10, compared with those of controls. Neonatal DES treatment reduced c-Jun immunoreactivity in the uterine epithelium on days 4 and 10, and increased the reaction in the stroma on day 4. These results suggested that the neonatal DES treatment induces permanent changes in the c-fos expression pattern independent of the postpuberal secretion of ovarian steroids. The changes in the expression of c-fos and c-jun protooncogenes, particularly during postnatal development, are likely to play important roles in the production of uterine abnormalities in the DES-mice.

  13. The temporal expression pattern of alpha-synuclein modulates olfactory neurogenesis in transgenic mice.

    Sebastian R Schreglmann

    Full Text Available Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS under the murine Thy1 (mThy1 promoter, a model known to have a particularly high tg expression associated with impaired olfaction.Survival of newly generated neurons (NeuN-positive in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.

  14. Comparison of gene expression changes induced by biguanides in db/db mice liver.

    Heishi, Masayuki; Hayashi, Koji; Ichihara, Junji; Ishikawa, Hironori; Kawamura, Takao; Kanaoka, Masaharu; Taiji, Mutsuo; Kimura, Toru


    Large-scale clinical studies have shown that the biguanide drug metformin, widely used for type 2 diabetes, to be very safe. By contrast, another biguanide, phenformin, has been withdrawn from major markets because of a high incidence of serious adverse effects. The difference in mode of action between the two biguanides remains unclear. To gain insight into the different modes of action of the two drugs, we performed global gene expression profiling using the livers of obese diabetic db/db mice after a single administration of phenformin or metformin at levels sufficient to cause a significant reduction in blood glucose level. Metformin induced modest expression changes, including G6pc in the liver as previously reported. By contrast, phenformin caused changes in expression level of many additional genes. We used a knowledge-based bioinformatic analysis to study the effects of phenformin. Differentially expressed genes identified in this study constitute a large gene network, which may be related to cell death, inflammation or wound response. Our results suggest that the two biguanides show a similar hypoglycemic effect in db/db mice, but phenformin induces a greater stress on the liver even a short time after a single administration. These findings provide a novel insight into the cause of the relatively high occurrence of serious adverse effect after phenformin treatment.

  15. Molecular and electrophysiological characterization of GFP-expressing CA1 interneurons in GAD65-GFP mice.

    Corette J Wierenga

    Full Text Available The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2% or vasoactive intestinal peptide (VIP; 15±2%, while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping expression of neuropeptide Y (42±3%, cholecystokinin (25±3%, calbindin (20±2% or calretinin (20±2%. Most of these subtypes (with the exception of calretinin-expressing interneurons target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.

  16. Faithful expression of the human 5q31 cytokine cluster intransgenic mice

    Lacy, Dee A.; Wang, Zhi-En; Symula, Derek J.; McArthur, CliffordJ.; Rubin, Edward M.; Frazer, Kelly A.; Locksley, Richard M.


    ILs 4,5, and 13, cardinal cytokines produced by Th2 cells,are coordinately expressed and clustered in the 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the5931cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human Il-4, IL-13, and Il-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2 inducing stimulus, and human Il-4 was generated after activation of NK T cells in vivo.Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 ctyokine genes in lymphocytes.

  17. Genome-wide gene expression profiling of the Angelman syndrome mice with Ube3a mutation.

    Low, Daren; Chen, Ken-Shiung


    Angelman syndrome (AS) is a human neurological disorder caused by lack of maternal UBE3A expression in the brain. UBE3A is known to function as both an ubiquitin-protein ligase (E3) and a coactivator for steroid receptors. Many ubiquitin targets, as well as interacting partners, of UBE3A have been identified. However, the pathogenesis of AS, and how deficiency of maternal UBE3A can upset cellular homeostasis, remains vague. In this study, we performed a genome-wide microarray analysis on the maternal Ube3a-deficient (Ube3a(m-/p+)) AS mouse to search for genes affected in the absence of Ube3a. We observed 64 differentially expressed transcripts (7 upregulated and 57 downregulated) showing more than 1.5-fold differences in expression (Pphenotype. We also show that the protein level of melanocortin 1 receptor (Mc1r) and nuclear receptor subfamily 4, group A, member 2 (Nr4a2) in the AS mice cerebellum is decreased relative to that of the wild-type mice. Consistent with this finding, expression of small-interfering RNA that targets Ube3a in P19 cells caused downregulation of Mc1r and Nr4a2, whereas overexpression of Ube3a results in the upregulation of Mc1r and Nr4a2. These observation help in providing insights into the genesis of neurodevelopmental phenotype of AS and highlight specific area for future research.

  18. Persistent neuronal Ube3a expression in the suprachiasmatic nucleus of Angelman syndrome model mice.

    Jones, Kelly A; Han, Ji Eun; DeBruyne, Jason P; Philpot, Benjamin D


    Mutations or deletions of the maternal allele of the UBE3A gene cause Angelman syndrome (AS), a severe neurodevelopmental disorder. The paternal UBE3A/Ube3a allele becomes epigenetically silenced in most neurons during postnatal development in humans and mice; hence, loss of the maternal allele largely eliminates neuronal expression of UBE3A protein. However, recent studies suggest that paternal Ube3a may escape silencing in certain neuron populations, allowing for persistent expression of paternal UBE3A protein. Here we extend evidence in AS model mice (Ube3a(m-/p+)) of paternal UBE3A expression within the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Paternal UBE3A-positive cells in the SCN show partial colocalization with the neuropeptide arginine vasopressin (AVP) and clock proteins (PER2 and BMAL1), supporting that paternal UBE3A expression in the SCN is often of neuronal origin. Paternal UBE3A also partially colocalizes with a marker of neural progenitors, SOX2, implying that relaxed or incomplete imprinting of paternal Ube3a reflects an overall immature molecular phenotype. Our findings highlight the complexity of Ube3a imprinting in the brain and illuminate a subpopulation of SCN neurons as a focal point for future studies aimed at understanding the mechanisms of Ube3a imprinting.

  19. Increased hippocampal Disrupted-In-Schizophrenia 1 expression in mice exposed prenatally to lead

    Yuanyuan You; Liguang Sun; Bo Peng; Yan Li; Songbin Ben; Shuang Gao


    Disrupted-In-Schizophrenia 1 is a susceptibility gene for schizophrenia and other psychiatric disorders.Developmental lead exposure can cause neurological disorders similar to hyperactivity disorder,dyslexia and schizophrenia. In the present study, we examined the impact of developmental lead exposure, administered in vitro and in vivo, on hippocampal Disrupted-In- Schizophrenia 1 expression. Our results show that in cultured hippocampal neurons, in vitro exposure to 0.1-10 μM lead, inhibited neurite growth and increased Disrupted-In-Schizophrenia 1 mRNA and protein expression dose-dependently. In addition, blood lead levels in mice were increased with increasing mouse maternal lead (0.01-1 mM) exposure. Hippocampal neurons from these mice showed a concomitant increase in Disrupted-In-Schizophrenia 1 mRNA and protein expression. Overall our findings suggest that in vivo and in vitro lead exposure increases Disrupted-In-Schizophrenia 1 expression in hippocampal neurons dose-dependently, and consequently may influence synapse formation in newborn neurons.

  20. Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice.

    Cissy Chenyi Zhou

    Full Text Available BACKGROUND: Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth. PRINCIPAL FINDINGS: By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa and adenosine deaminase (Ada, we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus. SIGNIFICANCE: In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.

  1. Expression of cartilage developmental genes in Hoxc8- and Hoxd4-transgenic mice.

    Claudia Kruger

    Full Text Available Hox genes encode transcription factors, which regulate skeletal patterning and chondrocyte differentiation during the development of cartilage, the precursor to mature bone. Overexpression of the homeobox transcription factors Hoxc8 and Hoxd4 causes severe cartilage defects due to delay in cartilage maturation. Matrix metalloproteinases (MMPs, bone morphogenetic proteins (BMPs and fibroblastic growth factors (FGFs are known to play important roles in skeletal development and endochondral bone formation and remodeling. In order to investigate whether these molecules are aberrantly expressed in Hoxc8- and/or Hoxd4-transgenic cartilage, we performed quantitative RT-PCR on chondrocytes from Hox-transgenic mice. Gene expression levels of Bmp4, Fgf8, Fgf10, Mmp9, Mmp13, Nos3, Timp3, Wnt3a and Wnt5a were altered in Hoxc8-transgenic chondrocytes, and Fgfr3, Ihh, Mmp8, and Wnt3a expression levels were altered in Hoxd4-transgenic chondrocytes, respectively. Notably, Wnt3a expression was elevated in Hoxc8- and reduced in Hoxd4-transgenic cartilage. These results suggest that both transcription factors affect cartilage maturation through different molecular mechanisms, and provide the basis for future studies into the role of these genes and possible interactions in pathogenesis of cartilage defects in Hoxc8- and Hoxd4-transgenic mice.

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

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


    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.

  3. Adult male mice conceived by in vitro fertilization exhibit increased glucocorticoid receptor expression in fat tissue.

    Simbulan, R K; Liu, X; Feuer, S K; Maltepe, E; Donjacour, A; Rinaudo, P


    Prenatal development is highly plastic and readily influenced by the environment. Adverse conditions have been shown to alter organ development and predispose offspring to chronic diseases, including diabetes and hypertension. Notably, it appears that the changes in glucocorticoid hormones or glucocorticoid receptor (GR) levels in peripheral tissues could play a role in the development of chronic diseases. We have previously demonstrated that in vitro fertilization (IVF) and preimplantation embryo culture is associated with growth alterations and glucose intolerance in mice. However, it is unknown if GR signaling is affected in adult IVF offspring. Here we show that GR expression is increased in inbred (C57Bl6/J) and outbred (CF-1× B6D2F1/J) blastocysts following in vitro culture and elevated levels are also present in the adipose tissue of adult male mice. Importantly, genes involved in lipolysis and triglyceride synthesis and responsive to GR were also increased in adipose tissue, indicating that increased GR activates downstream gene pathways. The promoter region of GR, previously reported to be epigenetically modified by perinatal manipulation, showed no changes in DNA methylation status. Our findings demonstrate that IVF results in a long-term change in GR gene expression in a sex- and tissue-specific manner. These changes in adipose tissues may well contribute to the metabolic phenotype in mice conceived by IVF.

  4. Expression of a structurally constrained von Willebrand factor variant triggers acute thrombotic thrombocytopenic purpura in mice.

    Morioka, Yoko; Casari, Caterina; Wohner, Nikolett; Cho, Sungyun; Kurata, Sachiko; Kitano, Ayumi; Christophe, Olivier D; Lenting, Peter J; Li, Renhao; Denis, Cécile V; Prévost, Nicolas


    Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease that presents with thrombocytopenia, disseminated thrombosis, hemolytic anemia, and organ dysfunction. The etiology of TTP has revealed that patients share a deficiency in plasma protease a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), the enzyme responsible for cleaving ultra-large von Willebrand factor (VWF) multimers into nonthrombogenic fragments. Therefore, existing TTP mouse models were developed by targeted disruption of the ADAMTS13 gene. ADAMTS13(-/-) mice are mostly asymptomatic in the absence of a trigger, as redundant proteases appear to take on VWF processing. As an alternative approach to creating one such model, we devised a strategy based on the expression of a cleavage-resistant VWF mutant in mice. The creation of a disulfide bond within the A2 domain of VWF was found to render VWF multimers resistant to proteolysis by plasma proteases under flow. Furthermore, mice expressing the murine VWF/p.S1494C-p.A1534C mutant present with symptoms characteristics of acute TTP such as thrombocytopenia, red cell shredding, accumulation of VWF-rich thrombi in the microvasculature, and advanced TTP symptoms such as renal dysfunction and splenomegaly. Because this model appears to faithfully emulate the pathophysiology of TTP, it should prove most useful in the study of microangiopathic diseases and their treatment. © 2014 by The American Society of Hematology.

  5. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

    Simonet, W S; DeRose, M L; Bucay, N; Nguyen, H Q; Wert, S E; Zhou, L; Ulich, T R; Thomason, A; Danilenko, D M; Whitsett, J A


    Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618921

  6. Long-term expression of periostin during the chronic stage of ischemic stroke in mice.

    Shimamura, Munehisa; Taniyama, Yoshiaki; Nakagami, Hironori; Katsuragi, Naruto; Wakayama, Kouji; Koriyama, Hiroshi; Kurinami, Hitomi; Tenma, Akiko; Tomioka, Hideki; Morishita, Ryuichi


    Periostin is an extracellular matrix glycoprotein and has various cellular effects. Previously, we demonstrated the neuroprotective effects of periostin during the acute stage of cerebral ischemia. However, its expression during the chronic stage remains unknown. Herein, we examined the expression of full-length periostin (periostin 1; Pn1) and its splicing variant lacking exon 17 (periostin 2; Pn2) during the 28 days following transient middle cerebral artery occlusion in mice. Real-time reverse transcription-PCR showed that the expression of Pn2 was dramatically upregulated between days 3 and 28, and the highest expression was observed on day 7. The expression of Pn1 was also increased, but delayed compared with Pn2. Immunohistochemistry showed that periostin was weakly expressed in reactive astrocytes in the peri-infarct region and in microglia/macrophages in infarct regions, on days 3 and 7. Periostin was also expressed around CD31-positive cells in both the peri-infarct and the sub-ventricular zone (SVZ) on days 3 and 7. SOX-2 positive cells, which are neural stem cells, also expressed periostin on day 7. The highest periostin immunoreactivity that occurred co-localized with collagen I and fibronectin in the peri-infarct region between days 7 and 28. Thus, the expression pattern of periostin mRNA was dependent on the splicing variant, and it continued to be expressed up to 28 days after cerebral ischemia. As periostin was expressed in various cells, such as reactive astrocytes/microglia, fibroblasts and neuronal progenitor cells, periostin might be associated with pathophysiology in post-ischemic inflammation and neurogenesis.

  7. Effects of expressing human T-cell leukemia virus type 1 (HTLV-I) oncoprotein Tax on DOK1, DOK2 and DOK3 gene expression in mice.

    Ohsugi, Takeo


    Transgenic mice expressing the tax gene from human T-cell leukemia virus type 1 (HTLV-I) genome developed T-cell leukemia or histiocytic sarcoma after at least 12 months. The transgenic mice showed low expression of the downstream of tyrosine kinase (DOK) family members, DOK1, DOK2 and DOK3, which were recently reported to be tumor suppressor genes. Mice showed low DOK2 expression at 5-6 months of age, before disease onset. The expression of DOK1 and DOK3 was not significantly reduced at any age tested. These results suggest that downregulation of DOK2 by the expression of the viral tax gene is the first step in the development of T-cell leukemia or histiocytic sarcoma.

  8. 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

    阴彦辉; 韦光辉; 李伟; 朱才业; 张亚妮; 杜立新; 曹文广; 李碧春


    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

  9. Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice.

    Jennifer O'Leary

    Full Text Available BACKGROUND: Williams-Beuren Syndrome (WBS is a neurodevelopmental disorder caused by a hemizygous deletion of a 1.5 Mb region on chromosome 7q11.23 encompassing 26 genes. One of these genes, GTF2IRD1, codes for a putative transcription factor that is expressed throughout the brain during development. Genotype-phenotype studies in patients with atypical deletions of 7q11.23 implicate this gene in the neurological features of WBS, and Gtf2ird1 knockout mice show reduced innate fear and increased sociability, consistent with features of WBS. Multiple studies have identified in vitro target genes of GTF2IRD1, but we sought to identify in vivo targets in the mouse brain. METHODOLOGY/PRINCIPAL FINDINGS: We performed the first in vivo microarray screen for transcriptional targets of Gtf2ird1 in brain tissue from Gtf2ird1 knockout and wildtype mice at embryonic day 15.5 and at birth. Changes in gene expression in the mutant mice were moderate (0.5 to 2.5 fold and of candidate genes with altered expression verified using real-time PCR, most were located on chromosome 5, within 10 Mb of Gtf2ird1. siRNA knock-down of Gtf2ird1 in two mouse neuronal cell lines failed to identify changes in expression of any of the genes identified from the microarray and subsequent analysis showed that differences in expression of genes on chromosome 5 were the result of retention of that chromosome region from the targeted embryonic stem cell line, and so were dependent upon strain rather than Gtf2ird1 genotype. In addition, specific analysis of genes previously identified as direct in vitro targets of GTF2IRD1 failed to show altered expression. CONCLUSIONS/SIGNIFICANCE: We have been unable to identify any in vivo neuronal targets of GTF2IRD1 through genome-wide expression analysis, despite widespread and robust expression of this protein in the developing rodent brain.

  10. Altered placental expression of PAPPA2 does not affect birth weight in mice

    Christians Julian K


    Full Text Available Abstract Background Pregnancy-associated plasma protein A2 (PAPPA2 is an insulin-like growth factor binding protein (IGFBP protease expressed in the placenta and upregulated in pregnancies complicated by pre-eclampsia. The mechanism linking PAPPA2 expression and pre-eclampsia and the consequences of altered PAPPA2 expression remain unknown. We previously identified PAPPA2 as a candidate gene for a quantitative trait locus (QTL affecting growth in mice and in the present study examined whether this QTL affects placental PAPPA2 expression and, in turn, placental or embryonic growth. Methods Using a line of mice that are genetically homogenous apart from a 1 megabase QTL region containing the PAPPA2 gene, we bred mice homozygous for alternate QTL genotypes and collected and weighed placentae and embryos at E12.5. We used quantitative RT-PCR to measure the mRNA levels of PAPPA2, as well as mRNA levels of IGFBP-5 (PAPPA2's substrate, and PAPPA (a closely related IGFBP protease to examine potential feedback and compensation effects. Western blotting was used to quantify PAPPA2 protein. Birth weight was measured in pregnancies allowed to proceed to parturition. Results PAPPA2 mRNA and protein expression levels in the placenta differed by a factor of 2.5 between genotypes, but we did not find a significant difference between genotypes in embryonic PAPPA2 mRNA levels. Placental IGFBP-5 and PAPPA mRNA expression levels were not altered in response to PAPPA2 levels, and we could not detect IGFBP-5 protein in the placenta by Western blotting. The observed difference in placental PAPPA2 expression had no significant effect on placental or embryonic mass at mid-gestation, birth weight or litter size. Conclusions Despite a significant difference between genotypes in placental PAPPA2 expression similar in magnitude to the difference between pre-eclamptic and normal placentae previously reported, we observed no difference in embryonic, placental or birth weight

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

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


    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

  12. Enhanced human immunodeficiency virus Type 1 expression and neuropathogenesis in knockout mice lacking Type I interferon responses.

    He, Hongxia; Sharer, Leroy R; Chao, Wei; Gu, Chao-Jiang; Borjabad, Alejandra; Hadas, Eran; Kelschenbach, Jennifer; Ichiyama, Koji; Do, Meilan; Potash, Mary Jane; Volsky, David J


    The roles of Type I interferon (IFN) in human immunodeficiency virus Type 1 (HIV-1) neuropathogenesis are poorly understood; both protective and deleterious effects of IFN signaling have been described. We used genetically modified mice deficient in the Type I IFN receptor (IFNRKO) to analyze the progress of HIV-1 brain infection and neuropathogenesis in the absence of IFN signaling. IFNRKO and wild-type (WT) mice on the 129xSv/Ev or C57BL/6 strain backgrounds were infected systemically with EcoHIV, a chimeric HIV-1 that productively infects mice. IFNRKO mice showed higher HIV-1 expression in spleen and peritoneal macrophages and greater virus infiltration into the brain compared to WT mice. Neuropathogenesis was studied by histopathological, immunohistochemical, immunofluorescence, and polymerase chain reaction analyses of brain tissues after the virus was inoculated into the brain by stereotaxic intracerebral injection. Both IFNRKO and WT mice showed readily detectable HIV-1 and brain lesions, including microglial activation, astrocytosis, and increased expression of genes coding for inflammatory cytokines and chemokines typical of human HIV-1 brain disease. Parameters of HIV-1 neuropathogenesis, including HIV-1 expression in microglia/macrophages, were significantly greater in IFNRKO than in WT mice. Our results show unequivocally that Type I IFN signaling and responses limit HIV-1 infection and pathogenesis in the brains of mice.

  13. Deletion of a coordinate regulator of type 2 cytokine expression in mice

    Mohrs, Markus; Blankespoor, Catherine M.; Wang, Zhi-En; Loots, Gaby G.; Hadeiba, Husein; Shinkai, Kanade; Rubin, Edward M.; Locksley, Richard M.


    Mechanisms underlying the differentiation of stable T helper subsets will be important in understanding how discrete types of immunity develop in response to different pathogens. An evolutionarily conserved {approx}400 base pair non-coding sequence in the IL-4/IL-13 intergenic region, designated CNS-1, was deleted in mice. The capacity to develop Th2 cells was compromised in vitro and in vivo in the absence of CNS-1. Despite the profound effect in T cells, mast cells from CNS-1-deleted mice maintained their capacity to produce IL-4. A T cell-specific element critical for optimal expression of type 2 cytokines may represent evolution of a regulatory sequence exploited by adaptive immunity.

  14. Reduced Mid1 expression and delayed neuromotor development in daDREAM transgenic mice

    Mara eDierssen


    Full Text Available DREAM (downstream regulatory element antagonist modulator is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. Previous work has shown a role for DREAM in cerebellar function regulating the expression of the sodium/calcium exchanger 3 (NCX3 in cerebellar granular neurons to control Ca2+ homeostasis and survival of these neurons. To achieve a global view of the genes regulated by DREAM in the cerebellum, we performed a genome-wide analysis in transgenic cerebellum expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM. Here we show that DREAM regulates the expression of the midline 1 (Mid1 gene early after birth. As a consequence, daDREAM mice exhibit a significant shortening of the rostro-caudal axis of the cerebellum and a severe delay in neuromotor development early after birth. Our results indicate a role for DREAM in cerebellar function.

  15. Effect of zearalenone on reproductive parameters and expression of selected testicular genes in mice.

    Zatecka, E; Ded, L; Elzeinova, F; Kubatova, A; Dorosh, A; Margaryan, H; Dostalova, P; Korenkova, V; Hoskova, K; Peknicova, J


    We tested the effect of two different concentrations (150μg/l and 0.15μg/l) of mycotoxin zearalenone (ZEA) on the reproductive parameters and expression of testicular genes in male mice. In adult males, no reduction of body or reproductive organ weight was observed, and the seminiferous tubules were morphologically normal with ongoing spermatogenesis. However, we found decreased sperm concentration, increase of morphologically abnormal spermatozoa and increased binding of apoptotic marker annexin V. This study was also focused on the evaluation of gene expression profiles of 28 genes playing important roles during the processes occurring in the testicular tissue. We detected changes in the expression of genes important for proper spermatogenesis. Surprisingly, we observed a stronger effect after exposure to the lower dose of ZEA.

  16. Pine pollen inhibits cell apoptosis-related protein expression in the cerebral cortex of mice with arsenic poisoning

    Yanhong Luo; Yaodong Wei; Taizhong Wang; Dongzhu Chen; Tiansheng Lu; Ruibo Wu; Keke Si


    Previous studies have demonstrated that pine pollen can inhibit cerebral cortical cell apoptosis in mice with arsenic poisoning. The present study sought to detect the influence of pine pollen on apoptosis-related proteins. Immunohistochemistry, western blotting and enzyme-linked immuno-sorbent assays were used to measure the levels of apoptosis-related proteins in the cerebral cortex of mice with arsenic poisoning. Results indicated that pine pollen suppressed cell apoptosis in the cerebral cortex of arsenic-poisoned mice by reducing Bax, Bcl-2 protein expression and increasing p53 protein expression.

  17. Global gene expression patterns in the post-pneumonectomy lung of adult mice

    Ingenito Edward P


    Full Text Available Abstract Background Adult mice have a remarkable capacity to regenerate functional alveoli following either lung resection or injury that exceeds the regenerative capacity observed in larger adult mammals. The molecular basis for this unique capability in mice is largely unknown. We examined the transcriptomic responses to single lung pneumonectomy in adult mice in order to elucidate prospective molecular signaling mechanisms used in this species during lung regeneration. Methods Unilateral left pneumonectomy or sham thoracotomy was performed under general anesthesia (n = 8 mice per group for each of the four time points. Total RNA was isolated from the remaining lung tissue at four time points post-surgery (6 hours, 1 day, 3 days, 7 days and analyzed using microarray technology. Results The observed transcriptomic patterns revealed mesenchymal cell signaling, including up-regulation of genes previously associated with activated fibroblasts (Tnfrsf12a, Tnc, Eln, Col3A1, as well as modulation of Igf1-mediated signaling. The data set also revealed early down-regulation of pro-inflammatory cytokine transcripts and up-regulation of genes involved in T cell development/function, but few similarities to transcriptomic patterns observed during embryonic or post-natal lung development. Immunohistochemical analysis suggests that early fibroblast but not myofibroblast proliferation is important during lung regeneration and may explain the preponderance of mesenchymal-associated genes that are over-expressed in this model. This again appears to differ from embryonic alveologenesis. Conclusion These data suggest that modulation of mesenchymal cell transcriptome patterns and proliferation of S100A4 positive mesenchymal cells, as well as modulation of pro-inflammatory transcriptome patterns, are important during post-pneumonectomy lung regeneration in adult mice.

  18. Gene Expression Profile in the Liver of BALB/c Mice Infected with Fasciola hepatica.

    Jose Rojas-Caraballo

    Full Text Available Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs. It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology.A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0, seven days post-infection (t7 and twenty-one days post-infection (t21.We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death.The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma

  19. The forkhead transcription factor, Foxd1, is necessary for pituitary luteinizing hormone expression in mice.

    Jason H Gumbel

    Full Text Available The pituitary gland regulates numerous physiological functions including growth, reproduction, temperature and metabolic homeostasis, lactation, and response to stress. Pituitary organogenesis is dependent on signaling factors that are produced in and around the developing pituitary. The studies described in this report reveal that the forkhead transcription factor, Foxd1, is not expressed in the developing mouse pituitary gland, but rather in the mesenchyme surrounding the pituitary gland, which is an essential source of signaling factors that regulate pituitary organogenesis. Loss of Foxd1 causes a morphological defect in which the anterior lobe of the pituitary gland protrudes through the cartilage plate that is developing ventral to the pituitary at embryonic days (e14.5, e16.5, and e18.5. The number of proliferating pituitary cells is increased at e14.5 and e16.5. Loss of Foxd1 also results in significantly decreased levels of Lhb expression at e18.5. This decrease in Lhb expression does not appear to be due to a change in the number of gonadotrope cells in the pituitary gland. Previous studies have shown that loss of the LIM homeodomain factor, Lhx3, which is activated by the FGF signaling pathway, results in loss of LH production. Although there is a difference in Lhb expression in Foxd1 null mice, the expression pattern of LHX3 is not altered in Foxd1 null mice. These studies suggest that Foxd1 is indirectly required for normal Lhb expression and cartilage formation.

  20. Gene expression related to oxidative stress in the heart of mice after intestinal ischemia

    Somaio Neto, Frederico; Ikejiri, Adauto Tsutomu; Bertoletto, Paulo Roberto; Chaves, José Carlos Bertoletto [Universidade Federal da Grande Dourados - UFGD, Dourados, MS (Brazil); Teruya, Roberto [Universidade Federal do Mato Grosso do Sul - UFMS, Campo Grande, MS (Brazil); Fagundes, Djalma José, E-mail:; Taha, Murched Omar [Universidade Federal de São Paulo - UNIFESP, São Paulo, SP (Brazil)


    Intestinal ischemia-reperfusion is a frequent clinical event associated to injury in distant organs, especially the heart. To investigate the gene expression of oxidative stress and antioxidant defense in the heart of inbred mice subjected to intestinal ischemia and reperfusion (IR). Twelve mice (C57BL / 6) were assigned to: IR Group (GIR) with 60 minutes of superior mesenteric artery occlusion followed by 60 minutes of reperfusion; Control Group (CG) which underwent anesthesia and laparotomy without IR procedure and was observed for 120 minutes. Intestine and heart samples were processed using the RT-qPCR / Reverse transcriptase-quantitative Polymerase Chain Reaction method for the gene expression of 84 genes related to oxidative stress and oxidative defense (Student's 't' test, p < 0.05). The intestinal tissue (GIR) was noted to have an up-regulation of 65 genes (74.71%) in comparison to normal tissue (CG), and 37 genes (44.04%) were hyper-expressed (greater than three times the threshold allowed by the algorithm). Regarding the remote effects of intestinal I/R in cardiac tissue an up-regulation of 28 genes (33.33%) was seen, but only eight genes (9.52%) were hyper-expressed three times above threshold. Four (7.14%) of these eight genes were expressed in both intestinal and cardiac tissues. Cardiomyocytes with smaller and pyknotic nuclei, rich in heterochromatin with rare nucleoli, indicating cardiac distress, were observed in the GIR. Intestinal I/R caused a statistically significant over expression of 8 genes associated with oxidative stress in remote myocardial tissue.

  1. Expression of PPARα modifies fatty acid effects on insulin secretion in uncoupling protein-2 knockout mice

    Chan Catherine B


    Full Text Available Abstract Aims/hypothesis In uncoupling protein-2 (UCP2 knockout (KO mice, protection of beta cells from fatty acid exposure is dependent upon transcriptional events mediated by peroxisome proliferator-activated receptor-α (PPARα. Methods PPARα expression was reduced in isolated islets from UCP2KO and wild-type (WT mice with siRNA for PPARα (siPPARα overnight. Some islets were also cultured with oleic or palmitic acid, then glucose stimulated insulin secretion (GSIS was measured. Expression of genes was examined by quantitative RT-PCR or immunoblotting. PPARα activation was assessed by oligonucleotide consensus sequence binding. Results siPPARα treatment reduced PPARα protein expression in KO and WT islets by >85%. In siPPARα-treated UCP2KO islets, PA but not OA treatment significantly decreased the insulin response to 16.5 mM glucose. In WT islets, siPPARα treatment did not modify GSIS in PA and OA exposed groups. In WT islets, PA treatment significantly increased UCP2 mRNA and protein expression. Both PA and OA treatment significantly increased PPARα expression in UCP2KO and WT islets but OA treatment augmented PPARα protein expression only in UCP2KO islets (p Conclusion These data show that the negative effect of saturated fatty acid on GSIS is mediated by PPARα/UCP2. Knockout of UCP2 protects beta-cells from PA exposure. However, in the absence of both UCP2 and PPARα even a short exposure (24 h to PA significantly impairs GSIS.

  2. E-cadherin expression in obesity-associated, Kras-initiated pancreatic ductal adenocarcinoma in mice.

    Stark, Alexander P; Chang, Hui-Hua; Jung, Xiaoman; Moro, Aune; Hertzer, Kathleen; Xu, Mu; Schmidt, Andrea; Hines, O Joe; Eibl, Guido


    The epithelial-mesenchymal transition (EMT) is critical in the development of invasive epithelial malignancies. EMT is accelerated by inflammation and results in decreased E-cadherin expression. Diet-induced obesity is an inflammatory state that accelerates pancreatic carcinogenesis; its effect on EMT and E-cadherin expression in the development of pancreatic ductal adenocarcinoma is unclear. Conditional Kras(G12D) mice were fed a control diet or a high-fat, high-calorie diet for 3 or 9 months (n = 10 each). Immunohistochemistry with anti-E-cadherin antibody was performed. E-cadherin expression was characterized by staining intensity, location, and proportion of positive cells. In vitro expression of E-cadherin and Slug in primary pancreatic intraepithelial neoplasia (PanIN) and cancer cells was determined by Western blot. The HFCD led to increased weight gain in both 3- (15.8 vs 5.6 g, P cancer, E-cadherin expression was aberrant, with loss of membranous staining and prominent cytoplasmic staining, associated with strong, cytoplasmic expression of β-catenin. In vitro expression of E-cadherin was greatest in primary PanIN cells, accompanied by absent Slug expression. Cancer cell lines demonstrated significantly decreased E-cadherin expression in the presence of upregulated Slug. Despite increased pancreatic inflammation and accelerated carcinogenesis, the high-fat, high-calorie diet did not induce changes in E-cadherin expression in PanIN lesions of all stages. Invasive lesions demonstrated aberrant cytoplasmic E-cadherin staining. Loss of normal membranous localization may reflect a functional loss of E-cadherin. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

    Yuan-Xiang Zhou; Maggie Yuk-Ting Lee; James Ming-Him Ng; Mee-Len Chye; Wing-Kin Yip; Sze-Yong Zee; Eric Lam


    AIM: To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SR1), to investigate the transgene expression and pE2 accumulation in plastids,and to evaluate the antigenic effect of the plastid-derived pE2 in mice.METHODS: Plastid-targeting vector pRB94-E2containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids,this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration .of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA.RESULTS: Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies.CONCLUSION: HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.

  4. Anesthesia for euthanasia influences mRNA expression in healthy mice and after traumatic brain injury.

    Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin; Thal, Serge C


    Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real

  5. Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia.

    Ratliff, Eric P; Gutierrez, Alejandra; Davis, Roger A


    Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.

  6. Constitutively Expressed αB—Crystallin in Heat Schock Transcription Factor 1 Knockout Mice Myocardium

    刘莉; 张洪慧; 丁国宪; 程蕴琳; 晏良军; BENJAMINIvorJ


    Objective:To invesligate the effects of heat shock transcription factor 1(HSF 1) gene on the constitutively expressed aB-Crystallin(αBC)in mice myocardium.Methods:The expression levels of constitutive αBC in HSF1 knockout(hsf1-/-) and HSF1 wild type (hsf1+/+) mice myocardium were evaluated by western blot and immunohistochemistry.Results:The αBC levels in hsf1-/- and hsf1+/_ were 68.42±4.16,100.00±7.58,respectively(P<0.05,cytosolic fraction),and 20.53±1.01,37.55±1.91,respectively(P<0.05,pellet fraction).The αBC signals decreased significantly in hsf1-/- myocardium when compared with those in hsf1+/+ myocardium stained with fluorescence immunohistochemistry.Conclusion.HSF1 is an important,but not the only factor,which mediates the constitutively expressed αBC.

  7. Cosmetics-triggered percutaneous remote control of transgene expression in mice

    Wang, Hui; Ye, Haifeng; Xie, Mingqi; Daoud El-Baba, Marie; Fussenegger, Martin


    Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies. PMID:25943548

  8. Inflammation enhances mu-opioid receptor transcription and expression in mice intestine.

    Pol, O; Alameda, F; Puig, M M


    Opioid receptors (ORs) and their mRNA are present in the central and peripheral nervous systems of mammals and in different peripheral tissues, including the gut. Using a model of croton oil-induced (CO) intestinal inflammation in mice, we have shown a 6-fold increase in the potency of the antitransit and antisecretory effects of mu-OR agonists, mediated by peripheral ORs. We postulate that the enhanced effects are mediated by an increase in the expression of intestinal OR. We used jejunum (stripped of the mucosal layer) from mice with CO-induced intestinal inflammation and, as control subjects, saline-treated animals (SS). We evaluated the quantity of mu-OR mRNA determined by a competitive reverse-transcriptase polymerase chain reaction; the levels of mu-OR protein by Western blot immunoassay, and the localization and number of cells expressing mu-OR using immunohistochemistry. The results show a significant increase of mu-OR mRNA (7.7-fold) and receptor protein (3-fold) during intestinal inflammation. Inflammation also induced a 64.3% increase in the number of neurons expressing mu-OR immunoreactivity in the myenteric plexus but not in the submucosal plexus. Our results show that intestinal inflammation enhances the transcription and translation of mu-OR mRNA, thus explaining the increased potency of mu-opioids during inflammation.

  9. Cosmetics-triggered percutaneous remote control of transgene expression in mice.

    Wang, Hui; Ye, Haifeng; Xie, Mingqi; Daoud El-Baba, Marie; Fussenegger, Martin


    Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies.

  10. Gene expression signatures that predict radiation exposure in mice and humans.

    Holly K Dressman


    Full Text Available BACKGROUND: The capacity to assess environmental inputs to biological phenotypes is limited by methods that can accurately and quantitatively measure these contributions. One such example can be seen in the context of exposure to ionizing radiation. METHODS AND FINDINGS: We have made use of gene expression analysis of peripheral blood (PB mononuclear cells to develop expression profiles that accurately reflect prior radiation exposure. We demonstrate that expression profiles can be developed that not only predict radiation exposure in mice but also distinguish the level of radiation exposure, ranging from 50 cGy to 1,000 cGy. Likewise, a molecular signature of radiation response developed solely from irradiated human patient samples can predict and distinguish irradiated human PB samples from nonirradiated samples with an accuracy of 90%, sensitivity of 85%, and specificity of 94%. We further demonstrate that a radiation profile developed in the mouse can correctly distinguish PB samples from irradiated and nonirradiated human patients with an accuracy of 77%, sensitivity of 82%, and specificity of 75%. Taken together, these data demonstrate that molecular profiles can be generated that are highly predictive of different levels of radiation exposure in mice and humans. CONCLUSIONS: We suggest that this approach, with additional refinement, could provide a method to assess the effects of various environmental inputs into biological phenotypes as well as providing a more practical application of a rapid molecular screening test for the diagnosis of radiation exposure.

  11. Gene expression analysis reveals early changes in several molecular pathways in cerebral malaria-susceptible mice versus cerebral malaria-resistant mice

    Grau Georges E


    Full Text Available Abstract Background Microarray analyses allow the identification and assessment of molecular signatures in whole tissues undergoing pathological processes. To better understand cerebral malaria pathogenesis, we investigated intra-cerebral gene-expression profiles in well-defined genetically cerebral malaria-resistant (CM-R and CM-susceptible (CM-S mice, upon infection by Plasmodium berghei ANKA (PbA. We investigated mouse transcriptional responses at early and late stages of infection by use of cDNA microarrays. Results Through a rigorous statistical approach with multiple testing corrections, we showed that PbA significantly altered brain gene expression in CM-R (BALB/c, and in CM-S (CBA/J and C57BL/6 mice, and that 327 genes discriminated between early and late infection stages, between mouse strains, and between CM-R and CM-S mice. We further identified 104, 56, 84 genes with significant differential expression between CM-R and CM-S mice on days 2, 5, and 7 respectively. The analysis of their functional annotation indicates that genes involved in metabolic energy pathways, the inflammatory response, and the neuroprotection/neurotoxicity balance play a major role in cerebral malaria pathogenesis. In addition, our data suggest that cerebral malaria and Alzheimer's disease may share some common mechanisms of pathogenesis, as illustrated by the accumulation of β-amyloid proteins in brains of CM-S mice, but not of CM-R mice. Conclusion Our microarray analysis highlighted marked changes in several molecular pathways in CM-S compared to CM-R mice, particularly at early stages of infection. This study revealed some promising areas for exploration that may both provide new insight into the knowledge of CM pathogenesis and the development of novel therapeutic strategies.

  12. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

    Chen, Huiyong; Choesang, Tenzin; Li, Huihui; Sun, Shuming; Pham, Petra; Bao, Weili; Feola, Maria; Westerman, Mark; Li, Guiyuan; Follenzi, Antonia; Blanc, Lionel; Rivella, Stefano; Fleming, Robert E; Ginzburg, Yelena Z


    Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

  13. Baclofen prevents the elevated plus maze behavior and BDNF expression during naloxone precipitated morphine withdrawal in male and female mice.

    Pedrón, Valeria T; Varani, André P; Balerio, Graciela N


    In previous studies we have shown that baclofen, a selective GABAB receptor agonist, prevents the somatic expression and reestablishes the dopamine and μ-opioid receptors levels, modified during naloxone-precipitated morphine withdrawal syndrome in male and female mice. There are no previous reports regarding sex differences in the elevated plus maze (EPM) and the expression of BDNF in morphine-withdrawn mice. The present study analyses the behavioral and biochemical variations during morphine withdrawal in mice of both sexes, and whether these variations are prevented with baclofen. Swiss-Webster albino prepubertal mice received morphine (2 mg/kg, i.p.) twice daily, for 9 consecutive days. On the 10th day, one group of morphine-treated mice received naloxone (opioid receptor antagonist; 6 mg/kg, i.p.) 1 h after the last dose of morphine to precipitate withdrawal. A second group received baclofen (2 mg/kg, i.p.) before naloxone administration. The EPM behavior was measured during 15 min after naloxone injection. The expression of BDNF-positive cells was determined by immunohistochemistry. Withdrawn male mice showed a higher percentage of time spent and number of entries to the open arms compared to withdrawn female mice. Baclofen prevented this behavior in both sexes. BDNF expression decreased in the AcbC, BNST, CeC, and CA3 of the hippocampus while increased in the BLA of morphine withdrawn male. Baclofen pretreatment prevented the BDNF expression observed in morphine withdrawn male mice in all the brain areas studied except in the CeC. Baclofen prevention of the EPM behavior associated to morphine withdrawal could be partially related to changes in BDNF expression.

  14. Influence of cell growth conditions and medium composition on EGFP photostability in live cells.

    Mamontova, Anastasia V; Bogdanov, Alexey M; Lukyanov, Konstantin A


    Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham's F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells. This property of Ham's F12 medium is associated with decreased concentrations of riboflavin and pyridoxine, and increased concentrations of FeSO4, cyanocobalamine, lipoic acid, hypoxanthine, and thymidine compared with DMEM. We also found that the rate of EGFP photobleaching strongly depends on cell growth conditions such as cell density and the concentration of serum. We conclude that both imaging medium composition and the physiological state of the cells can strongly affect the photostability of fluorescent proteins. Thus, accurate comparison of the photostabilities of fluorescent proteins should be performed only in side-by-side analysis in identical cell growth conditions and media.

  15. Differentially expressed microRNAs at different stages of atherosclerosis in ApoE-deficient mice

    SHAN Zhen; YAO Chen; LI Zi-lun; TENG Yuan; LI Wen; WANG Jin-song; YE Cai-sheng


    Background Atherosclerosis is the primary cause of cardiovascular disease,carotid artery disease,and peripheral vascular disease.However,it is hard to obtain human arterial tissue at different stages of atherosclerosis for a systematic study.The ApoE-deficient (ApoE 1-) mice predictably develop spontaneous atherosclerotic plaques with numerous features similar to the human lesions and contain nearly the entire spectrum of lesions observed during atherogenesis in humans.MicroRNA expression profiles at different stages of atherosclerosis in ApoE-deficient mice were screened to find out the differentially expressed microRNAs.Methods ApoE-deficient mice were euthanized at 4,8,and 20 weeks of age and divided into three groups according to the three time points,including groups A4 (fed a Western-type diet for 0 week),A8 (fed a Western-type diet for 4 weeks),and A20 (fed a Western-type diet for 16 weeks).Atherosclerotic lesions were analyzed.Fifteen aortas were collected and combined into three pools (five aortas in one pool) in each group.MicroRNA microarray analysis was replicated thrice in each group.The threshold of fold change ≥2.0 was used to screen up or down-regulated microRNAs.Differentially expressed microRNAs were subsequently verified with quantitative real-time polymerase chain reaction.Those increasingly up or down-regulated microRNAs during the progression of atherosclerosis were selected.Results Atherosclerotic lesions first appeared in the aortic arch in group A8.Severe atherosclerotic lesions were observed in group A20.In group A8,seven MicroRNAs were up-regulated while two were down-regulated.In group A20,15 microRNAs were up-regulated while two were down-regulated.miR-34a-Sp and miR-497-5p were increasingly up-regulated,while miR-434-3p was progressively down-regulated when atherosclerosis progressed.Conclusions In this study,we described that microRNAs are differentially expressed at different stages of atherosclerosis in ApoE-deficient mice

  16. Production and characterization of transgenic mice systemically expressing endo-beta-galactosidase C.

    Watanabe, Satoshi; Misawa, Masako; Matsuzaki, Takashi; Sakurai, Takayuki; Muramatsu, Takashi; Yokomine, Taka-Aki; Sato, Masahiro


    The alphaGal epitope (Galalpha1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alphaGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alphaGal epitope by cleaving the Galbeta1-4GlcNAc linkage in the Galalpha1-3Galbeta1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the alphaGal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in beta1,4-galactosyltransferase 1 (beta4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation.

  17. Preventive DNA vaccination against CEA-expressing tumors with anti-idiotypic scFv6.C4 DNA in CEA-expressing transgenic mice.

    Denapoli, Priscila M A; Zanetti, Bianca F; Dos Santos, Adara A; de Moraes, Jane Z; Han, Sang W


    Carcinoembryonic antigen (CEA) is expressed during embryonic life and in low level during adult life. Consequently, the CEA is recognized by the immune system as a self-antigen and thus CEA-expressing tumors are tolerated. Previously, we constructed a single chain variable fragment using the 6.C4 (scFv6.C4) hybridoma cell line, which gave rise to antibodies able to recognize CEA when C57/Bl6 mice were immunized. Here, the scFv6.C4 ability to prevent the CEA-expressing tumor growth was assessed in CEA-expressing transgenic mice CEA2682. CEA2682 mice immunized with the scFv6.C4 expressing plasmid vector (uP/PS-scFv6.C4) by electroporation gave rise to the CEA-specific AB3 antibody after the third immunization. Sera from immunized mice reacted with CEA-expressing human colorectal cell lines CO112, HCT-8, and LISP-1, as well as with murine melanoma B16F10 cells expressing CEA (B16F10-CEA). Cytotoxic T lymphocytes (CTL) from uP/PS-scFv6.C4 immunized mice lysed B16F10-CEA (56.7%) and B16F10 expressing scFv6.C4 (B16F10-scFv6.C4) (46.7%) cells, against CTL from uP-immunized mice (10%). After the last immunization, 5 × 10(5) B16F10-CEA cells were injected into the left flank. All mice immunized with the uP empty vector died within 40 days, but uP/PS-scFv6.C4 vaccinated mice (40%) remained free of tumor for more than 100 days. Splenocytes obtained from uP/PS-scFv6.C4 vaccinated mice showed higher T-cell proliferative activity than those from uP vaccinated mice. Collectively, DNA vaccination with the uP-PS/scFv6.C4 plasmid vector was able to give rise to specific humoral and cellular responses, which were sufficient to retard growth and/or eliminate the injected B16F10-CEA cells.

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

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


    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

  19. Defective Gonadotropin-Dependent Ovarian Folliculogenesis and Granulosa Cell Gene Expression in Inhibin-Deficient Mice

    Nagaraja, Ankur K.; Middlebrook, Brooke S.; Rajanahally, Saneal; Myers, Michelle; Li, Qinglei; Matzuk, Martin M.; Pangas, Stephanie A.


    Inhibin-α knockout (Inha−/−) female mice develop sex cord-stromal ovarian cancer with complete penetrance and previous studies demonstrate that the pituitary gonadotropins (FSH and LH) are influential modifiers of granulosa cell tumor development and progression in inhibin-deficient females. Recent studies have demonstrated that Inha−/− ovarian follicles develop precociously to the early antral stage in prepubertal mice without any increase in serum FSH. These studies suggest that in the absence of inhibins, granulosa cells differentiate abnormally and thus at sexual maturity may undergo an abnormal response to gonadotropin signaling contributing to tumor development. To test this hypothesis, we stimulated immature wild-type and Inha−/− female mice with gonadotropin analogs prior to tumor formation and subsequently examined gonadotropin-induced ovarian follicle development as well as preovulatory and human chorionic gonadotropin-induced gene expression changes in granulosa cells. We find that at 3 wk of age, inhibin-deficient ovaries do not show further antral development or undergo cumulus expansion. In addition, there are widespread alterations in the transcriptome of gonadotropin-treated Inha−/− granulosa cells, with significant changes in genes involved in extracellular matrix and cell-cell communication. These data indicate the gonadotropins initiate an improper program of cell differentiation prior to tumor formation in the absence of inhibins. PMID:20739397

  20. Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

    Kerr, D E; Plaut, K; Bramley, A J; Williamson, C M; Lax, A J; Moore, K; Wells, K D; Wall, R J


    Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

  1. Brain beta-amyloid accumulation in transgenic mice expressing mutant superoxide dismutase 1.

    Turner, Bradley J; Li, Qiao-Xin; Laughton, Katrina M; Masters, Colin L; Lopes, Elizabeth C; Atkin, Julie D; Cheema, Surindar S


    Oxidative stress is implicated in both the deposition and pathogenesis of beta-amyloid (Abeta) protein in Alzheimer's disease (AD). Accordingly, overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) in neuronal cells and transgenic AD mice reduces Abeta toxicity and accumulation. In contrast, mutations in SOD1 associated with amyotrophic lateral sclerosis (ALS) confer enhanced pro-oxidative enzyme activities. We therefore examined whether ALS-linked mutant SOD1 overexpression in motor neuronal cells or transgenic ALS mice modulates Abeta toxicity or its accumulation in the brain. Aggregated, but not freshly solubilised, substrate-bound Abeta peptides induced degenerative morphology and cytotoxicity in motor neuron-like NSC-34 cells. Transfection of NSC-34 cells with human wild-type SOD1 attenuated Abeta-induced toxicity, however this neuroprotective effect was also observed for ALS-linked mutant SOD1. Analysis of the cerebral cortex, brainstem, cerebellum and olfactory bulb from transgenic SOD1G93A mice using enzyme-linked immunosorbent assay of acid-guanidine extracts revealed age-dependent elevations in Abeta levels, although not significantly different from wild-type mouse brain. In addition, brain amyloid protein precursor (APP) levels remained unaltered as a consequence of mutant SOD1 expression. We therefore conclude that mutant SOD1 overexpression promotes neither Abeta toxicity nor brain accumulation in these ALS models.

  2. Transgenic mice expressing human glucocerebrosidase variants: utility for the study of Gaucher disease.

    Sanders, Angela; Hemmelgarn, Harmony; Melrose, Heather L; Hein, Leanne; Fuller, Maria; Clarke, Lorne A


    Gaucher disease is an autosomal recessively inherited storage disorder caused by deficiency of the lysosomal hydrolase, acid β-glucosidase. The disease manifestations seen in Gaucher patients are highly heterogeneous as is the responsiveness to therapy. The elucidation of the precise factors responsible for this heterogeneity has been challenging as the development of clinically relevant animal models of Gaucher disease has been problematic. Although numerous murine models for Gaucher disease have been described each has limitations in their specific utility. We describe here, transgenic murine models of Gaucher disease that will be particularly useful for the study of pharmacological chaperones. We have produced stable transgenic mouse strains that individually express wild type, N370S and L444P containing human acid β-glucosidase and show that each of these transgenic lines rescues the lethal phenotype characteristic of acid β-glucosidase null mice. Both the N370S and L444P transgenic models show early and progressive elevations of tissue sphingolipids with L444P mice developing progressive splenic Gaucher cell infiltration. We demonstrate the potential utility of these new transgenic models for the study of Gaucher disease pathogenesis. In addition, since these mice produce only human enzyme, they are particularly relevant for the study of pharmacological chaperones that are specifically targeted to human acid β-glucosidase and the common mutations underlying Gaucher disease.

  3. Expression Profiling after Prolonged Experimental Febrile Seizures in Mice Suggests Structural Remodeling in the Hippocampus.

    Bart C Jongbloets

    Full Text Available Febrile seizures are the most prevalent type of seizures among children up to 5 years of age (2-4% of Western-European children. Complex febrile seizures are associated with an increased risk to develop temporal lobe epilepsy. To investigate short- and long-term effects of experimental febrile seizures (eFS, we induced eFS in highly febrile convulsion-susceptible C57BL/6J mice at post-natal day 10 by exposure to hyperthermia (HT and compared them to normotherm-exposed (NT mice. We detected structural re-organization in the hippocampus 14 days after eFS. To identify molecular candidates, which entrain this structural re-organization, we investigated temporal changes in mRNA expression profiles eFS 1 hour to 56 days after eFS. We identified 931 regulated genes and profiled several candidates using in situ hybridization and histology at 3 and 14 days after eFS. This is the first study to report genome-wide transcriptome analysis after eFS in mice. We identify temporal regulation of multiple processes, such as stress-, immune- and inflammatory responses, glia activation, glutamate-glutamine cycle and myelination. Identification of the short- and long-term changes after eFS is important to elucidate the mechanisms contributing to epileptogenesis.

  4. Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones.

    Matsuda, Atsushi; Shao, Lin; Boulanger, Jerome; Kervrann, Charles; Carlton, Peter M; Kner, Peter; Agard, David; Sedat, John W


    Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10-30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.

  5. Subnormal albumin gene expression is associated with weight loss in immunodeficient/DNA-repair-deficient wasted mice

    Libertin, C.R. [Loyola Univ., Maywood, IL (United States). Stritch School of Medicine; Weaver, P.; Woloschak, G.E. [Loyola Univ., Maywood, IL (United States). Stritch School of Medicine]|[Argonne National Lab., IL (United States); Mobarhan, S. [Loyola Univ., Maywood, IL (United States). Stritch School of Medicine


    Mice bearing the autosomal recessive mutation wst express a disease syndrome of immunodeficiency, neurologic dysfunction, and increased sensitivity to the killing effects of ionizing radiation. The mice were originally characterized as ``wasted`` because of their dramatic weight loss that begins at 21 days of age and progresses until death at 28-32 days of age. Because of the reported association between abnormal liver status and weight loss, we examined expression of a variety of liver-specific genes in wst/wst 10 mice relative to littermate (wst/{center_dot}) and parental strain (BCF{sub 1}) controls. Interestingly, the results revealed a greater than 67% reduction in albumin mRNA expression in livers derived from wst/wst mice relative to both controls. Expression of alpha-fetoprotein as well as a variety of other liver-specific genes (secretory component, metallothionein, cytochrome P{sub 1}450, transferrin receptor, tumor necrosis factor, and Ia antigen) was unaffected. These results suggest a relationship between low albumin expression and wasting syndromes in mice. In addition, we believe that our data suggest the wasted mouse as a unique model for subnormal albumin expression in humans.

  6. Expression of CD28 and CTLA4 on T Cells in Bone Morrow of Immune-mediated Aplastic Anemia Mice


    To investigate the expression and significance of CD28 and CTLA4 on T cells in bone marrow of aplastic anemia (AA) mice, in vitro bone marrow mononuclear cells (BMMNCs) were activated through being incubated with PHA (15μg/mL). The expression of CD28 and CTLA4 on T cells incubated with or without PHA was detected by two-color flow cytometry. The expression of CD28 and CTLA4 was significantly increased after PHA stimulation. In the AA mice, the expression of CD28 with or without PHA stimulation was both higher than that in the normal mice (both P<0.01), but the expression of CTLA4 with or without PHA stimulation showed no significant difference in comparison to that in the normal mice (both P>0.05). In the AA mice, there were more activation and activated potential of T cells than the normal, and the abnormal expression of CD28 and CTLA4 may participate in immunological disorder mediated by T cells.

  7. Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice.

    Qu, Yine; Zhang, Qiuyang; Ma, Siqi; Liu, Sen; Chen, Zhiquan; Mo, Zhongfu; You, Zongbing


    The functions of interleukin-17A (IL-17A) in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice) or a high-fat diet (n = 6, obese mice) for 30 weeks. Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice.

  8. Transcriptome profiling reveals divergent expression shifts in brown and white adipose tissue from long-lived GHRKO mice.

    Stout, Michael B; Swindell, William R; Zhi, Xu; Rohde, Kyle; List, Edward O; Berryman, Darlene E; Kopchick, John J; Gesing, Adam; Fang, Yimin; Masternak, Michal M


    Mice lacking the growth hormone receptor (GHRKO) exhibit improved lifespan and healthspan due to loss of growth hormone signaling. Both the distribution and activity of brown and white adipose tissue (BAT and WAT) are altered in GHRKO mice, but the contribution of each tissue to age-related phenotypes has remained unclear. We therefore used whole-genome microarrays to evaluate transcriptional differences in BAT and WAT depots between GHRKO and normal littermates at six months of age. Our findings reveal a unique BAT transcriptome as well as distinctive responses of BAT to Ghr ablation. BAT from GHRKO mice exhibited elevated expression of genes associated with mitochondria and metabolism, along with reduced expression of genes expressed by monocyte-derived cells (dendritic cells [DC] and macrophages). Largely the opposite was observed in WAT, with increased expression of DC-expressed genes and reduced expression of genes associated with metabolism, cellular respiration and the mitochondrial inner envelope. These findings demonstrate divergent response patterns of BAT and WAT to loss of GH signaling in GHRKO mice. These patterns suggest both BAT and WAT contribute in different ways to phenotypes in GHRKO mice, with Ghr ablation blunting inflammation in BAT as well as cellular metabolism and mitochondrial biogenesis in WAT.

  9. Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice

    Yine Qu


    Full Text Available The functions of interleukin-17A (IL-17A in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice or a high-fat diet (n = 6, obese mice for 30 weeks. Subcutaneous adipose tissue (SAT and visceral adipose tissue (VAT were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice.

  10. Muscle-directed gene therapy for phenylketonuria (PKU): Development of transgenic mice with muscle-specific phenylalanine hydroxylase expression

    Harding, C.O.; Messing, A.; Wolff, J.A. [Univ. of Wisconsin, Madison, WI (United States)


    Phenylketonuria (PKU) is an attractive target for gene therapy because of shortcomings in current therapy including lifelong commitment to a difficult and expensive diet, persistent mild cognitive deficits in some children despite adequate dietary therapy, and maternal PKU syndrome. Phenylalanine hydroxylase (PAH) is normally expressed only in liver, but we propose to treat PKU by introducing the gene for PAH into muscle. In order to evaluate both the safety and efficacy of this approach, we have a developed a trangenic mouse which expresses PAH in both cardiac and skeletal muscle. The transgene includes promoter and enhancer sequences from the mouse muscle creatine kinase (MCK) gene fused to the mouse liver PAH cDNA. Mice which have inherited the transgene are healthy, active, and do not exhibit any signs of muscle weakness or wasting. Ectopic PAH expression in muscle is not detrimental to the health, neurologic function, or reproduction of the mice. Pah{sup enu2} hyperphenylalaninemic mice, a model of human PAH deficiency, bred to carry the transgene have substantial PAH expression in cardiac and skeletal muscle but none in liver. Muscle PAH expression alone does not complement the hyperphenylalaninemic phenotype of Pah{sup enu2} mice. However, administration of reduced tetrahydrobiopterin to transgenic Pah{sup enu2} mice is associated with a 25% mean decrease in serum phenylalanine levels. We predict that ectopic expression of PAH in muscle along with adequate muscle supplies of reduced biopterin cofactor will decrease hyperphenylalaninemia in PKU.

  11. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    Luca Caracciolo

    Full Text Available Kainic acid (KA binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/- mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/- mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/- mice compared to wild type (WT mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/- mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/- compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/- mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/- mice. After KA exposure, COX-2(-/- mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6, inducible nitric oxide synthase (iNOS, microglia (CD11b and astrocyte (GFAP. Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/- mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  12. Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.

    Püschel, A W; Gruss, P; Westerfield, M


    Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain includin