Displaying publications 1 - 20 of 31 in total

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  1. Futra D, Heng LY, Ahmad A, Surif S, Ling TL
    Sensors (Basel), 2015 May 28;15(6):12668-81.
    PMID: 26029952 DOI: 10.3390/s150612668
    A fluorescence-based fiber optic toxicity biosensor based on genetically modified Escherichia coli (E. coli) with green fluorescent protein (GFP) was developed for the evaluation of the toxicity of several hazardous heavy metal ions. The toxic metals include Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III). The optimum fluorescence excitation and emission wavelengths of the optical biosensor were 400 ± 2 nm and 485 ± 2 nm, respectively. Based on the toxicity observed under optimal conditions, the detection limits of Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III) that can be detected using the toxicity biosensor were at 0.04, 0.32, 0.46, 2.80, 100, 250, 400, 720 and 2600 μg/L, respectively. The repeatability and reproducibility of the proposed biosensor were 3.5%-4.8% RSD (relative standard deviation) and 3.6%-5.1% RSD (n = 8), respectively. The biosensor response was stable for at least five weeks, and demonstrated higher sensitivity towards metal toxicity evaluation when compared to a conventional Microtox assay.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  2. Vakhshiteh F, Allaudin ZN, Lila MA, Abbasiliasi S, Ajdari Z
    Mol Biotechnol, 2015 Jan;57(1):75-83.
    PMID: 25218408 DOI: 10.1007/s12033-014-9803-8
    Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  3. Narayanan K, Lee CW, Radu A, Sim EU
    Anal Biochem, 2013 Aug 15;439(2):142-4.
    PMID: 23608053 DOI: 10.1016/j.ab.2013.04.010
    Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism*
  4. Li H, Yang C, Yusoff NM, Yahaya BH, Lin J
    Neuroscience, 2017 09 01;358:269-276.
    PMID: 28687312 DOI: 10.1016/j.neuroscience.2017.06.053
    Few researchers have investigated the direction of commissural axon projections on the contralateral side of the vertebrate embryonic spinal cord, especially for comparison between its different regions. In this study, pCAGGS-GFP plasmid expression was limited to different regions of the chicken embryonic spinal cord (cervical, anterior limb, anterior thorax, posterior thorax and posterior limb) at E3 using in ovo electroporation with modified electrodes and optimal electroporation conditions. Then open-book technique was performed at E6 to analyze the direction of axon projections in different spinal cord regions. The results show that in the five investigated regions, most axons projected rostrally after crossing the floor plate while a minority projected caudally. And there was a significant difference between the rostral and caudal projection quantities (P<0.01). The ratio of rostral and caudal projections was significantly different between the five investigated regions (P<0.05), except between the cervical region and the anterior limb (P>0.05). The projections were most likely to be rostral for the posterior limb followed by the posterior thorax, cervical region, anterior limb and anterior thorax. Our data for the direction of the commissural axon projections will be helpful in the future analyses of axon projection mechanisms and spinal cord-brain circuit formation.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  5. Abdul Halim NS, Fakiruddin KS, Ali SA, Yahaya BH
    Int J Mol Sci, 2014;15(9):15044-60.
    PMID: 25162825 DOI: 10.3390/ijms150915044
    Mesenchymal stem cells (MSCs) hold tremendous potential for therapeutic use in stem cell-based gene therapy. Ex vivo genetic modification of MSCs with beneficial genes of interest is a prerequisite for successful use of stem cell-based therapeutic applications. However, genetic manipulation of MSCs is challenging because they are resistant to commonly used methods to introduce exogenous DNA or RNA. Herein we compared the effectiveness of several techniques (classic calcium phosphate precipitation, cationic polymer, and standard electroporation) with that of microporation technology to introduce the plasmid encoding for angiopoietin-1 (ANGPT-1) and enhanced green fluorescent protein (eGFP) into human adipose-derived MSCs (hAD-MSCs). The microporation technique had a higher transfection efficiency, with up to 50% of the viable hAD-MSCs being transfected, compared to the other transfection techniques, for which less than 1% of cells were positive for eGFP expression following transfection. The capability of cells to proliferate and differentiate into three major lineages (chondrocytes, adipocytes, and osteocytes) was found to be independent of the technique used for transfection. These results show that the microporation technique is superior to the others in terms of its ability to transfect hAD-MSCs without affecting their proliferation and differentiation capabilities. Therefore, this study provides a foundation for the selection of techniques when using ex vivo gene manipulation for cell-based gene therapy with MSCs as the vehicle for gene delivery.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  6. Ngai SC, Rosli R, Nordin N, Veerakumarasivam A, Abdullah S
    Gene, 2012 May 1;498(2):231-6.
    PMID: 22366305 DOI: 10.1016/j.gene.2012.01.071
    Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14 days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  7. Molouki A, Hsu YT, Jahanshiri F, Abdullah S, Rosli R, Yusoff K
    Virol J, 2011;8:385.
    PMID: 21810274 DOI: 10.1186/1743-422X-8-385
    The underlying mechanisms by which Newcastle disease virus (NDV) kills cancer cells are still unclear. Recent discoveries have shown that many viruses contain Bcl-2 homology-like domains which enabled their interaction with Bcl-2 family members, and thereby accounting for their virulence and pathogenicity. Alignment of the protein sequences of Malaysian strain of NDV, known as AF2240, with those from members of the human Bcl-2 family showed many similar regions; most notably we found that its matrix (AF2240-M) protein, large (AF2240-L) protein and fusion (AF2240-F) protein all contain BH3-like regions. In addition, there are BH1-like domains in these proteins, where AF2240-F and Mcl-1 share 55% identity within this region. To further investigate our hypothesis that the presence of the BH3-like domains in these proteins may convey cytotoxicity, AF2240-M and AF2240-F genes were cloned into pFLAG and pEGFP.N2 vectors and transfected into HeLa cells. The expression of these constructs promoted cell death. As shown by flow cytometry, AF2240-M protein with deleted BH3-like region showed five-fold decrease in apoptosis. Moreover, the construct containing the N-terminal of AF2240-M showed nearly the same cell death rate as to that of the full-length protein, strongly suggesting that the BH3-like domain within this protein participates in promoting cell death. Moreover, AF2240-M transfection promoted Bax redistribution to mitochondria. Therefore, to determine whether there is any direct interaction between NDV viral proteins with some members of the Bcl-2 family, various constructs were co-transfected into HeLa cells. Co-immunoprecipitation trials showed that the AF2240-M indeed directly interacted with Bax protein via its BH3-domain, as the mutant proteins failed to interact with Bax. AF2240-F failed to interact with any of the tested proteins, although Bcl-XL slowed down the rate of cell death caused by this construct by nearly five-fold. In a parallel experiment, the level of expression of endogenous Bax and Bcl-2 after infection of HeLa cells with NDV was assessed by qRT-PCR, but no statistically significant change was observed. Consequently, the Bax/Bcl-2 ratio at the mRNA level did not alter. Overall, our study has shed additional light into the mechanisms by which NDV induces apoptosis.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  8. Khetawat D, Broder CC
    Virol J, 2010 Nov 12;7:312.
    PMID: 21073718 DOI: 10.1186/1743-422X-7-312
    BACKGROUND: Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.

    RESULTS: Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.

    CONCLUSIONS: Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.

    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  9. Das Gupta M, Chan SK, Monteiro A
    PLoS One, 2015;10(7):e0132882.
    PMID: 26173066 DOI: 10.1371/journal.pone.0132882
    Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  10. Seah TC, Tay YL, Tan HK, Muhammad TS, Wahab HA, Tan ML
    Int. J. Toxicol., 2015 08 12;34(5):454-68.
    PMID: 26268769 DOI: 10.1177/1091581815599335
    A cell-based assay to measure cytochrome P450 3A4 (CYP3A4) induction was developed to screen for potential CYP3A4 inducers. This 96-well format assay utilizes HepG2 cells transfected with a gene construct of CYP3A4 proximal promoter linked to green fluorescence protein (GFP) gene, and the expression of the GFP is then measured quantitatively. Bergamottin at 5 to 25 µmol/L produced low induction relative to the positive control. Both curcumin and lycopene were not found to affect the expression of GFP, suggesting no induction properties toward CYP3A4. Interestingly, resveratrol produced significant induction from 25 µmol/L onward, which was similar to omeprazole and may warrant further studies. In conclusion, the present study demonstrated that this cell-based assay can be used as a tool to evaluate the potential CYP3A4 induction properties of compounds. However, molecular docking data have not provided satisfactory pointers to differentiate between CYP3A4 inducers from noninducers or from inhibitors, more comprehensive molecular screening may be indicated.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  11. Marin-Mogollon C, van de Vegte-Bolmer M, van Gemert GJ, van Pul FJA, Ramesar J, Othman AS, et al.
    Sci Rep, 2018 10 08;8(1):14902.
    PMID: 30297725 DOI: 10.1038/s41598-018-33236-x
    Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  12. Teow SY, Liew K, Che Mat MF, Marzuki M, Abdul Aziz N, Chu TL, et al.
    BMC Biotechnol., 2019 06 14;19(1):34.
    PMID: 31200673 DOI: 10.1186/s12896-019-0528-4
    BACKGROUND: In vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC).

    RESULTS: Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated.

    CONCLUSIONS: XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.

    Matched MeSH terms: Green Fluorescent Proteins/metabolism*
  13. Zaborowski MP, Lee K, Na YJ, Sammarco A, Zhang X, Iwanicki M, et al.
    Cell Rep, 2019 04 02;27(1):255-268.e6.
    PMID: 30943406 DOI: 10.1016/j.celrep.2019.03.003
    Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  14. Abdul Mutalib NE, Mat Isa N, Alitheen NB, Song AA, Rahim RA
    Plasmid, 2014 May;73:26-33.
    PMID: 24780699 DOI: 10.1016/j.plasmid.2014.04.003
    Plasmid DNAs isolated from lactic acid bacteria (LAB) such as Lactococcus lactis (L. lactis) has been gaining more interests for its positive prospects in genetic engineering-related applications. In this study, the lactococcal plasmid, pNZ8048 was modified so as to be able to express multiple genes in the eukaryotic system. Therefore, a cassette containing an internal ribosome entry site (IRES) was cloned between VP2 gene of a very virulent infectious bursal disease (vvIBDV) UPM 04190 of Malaysian local isolates and the reporter gene, green fluorescent protein (GFP) into pNZ:CA, a newly constructed derivative of pNZ8048 harboring the cytomegalovirus promoter (Pcmv) and polyadenylation signal. The new bicistronic vector, denoted as pNZ:vig was subjected to in vitro transcription/translation system followed by SDS-PAGE and Western blot analysis to rapidly verify its functionality. Immunoblotting profiles showed the presence of 49 and 29kDa bands that corresponds to the sizes of the VP2 and GFP proteins respectively. This preliminary result shows that the newly constructed lactococcal bicistronic vector can co-express multiple genes in a eukaryotic system via the IRES element thus suggesting its feasibility to be used for transfection of in vitro cell cultures and vaccine delivery.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism*
  15. Yang C, Li X, Li S, Chai X, Guan L, Qiao L, et al.
    J Cell Mol Med, 2019 03;23(3):1813-1826.
    PMID: 30565384 DOI: 10.1111/jcmm.14080
    Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)-positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30-mm culture dish and 1 ml of slice culture media was added. We show that during serum-free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured-tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum-free medium cultured-tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism*
  16. Hang CY, Kitahashi T, Parhar IS
    J. Comp. Neurol., 2014 Dec 1;522(17):3847-60.
    PMID: 25043553 DOI: 10.1002/cne.23645
    In addition to vision, light information is used to regulate a range of animal physiology. Such nonimage-forming functions of light are mediated by nonvisual photoreceptors expressed in distinct neurons in the retina and the brain in most vertebrates. A nonvisual photoreceptor vertebrate ancient long opsin (VAL-opsin) possesses two functional isoforms in the zebrafish, encoded by valopa and valopb, which has received little attention. To delineate the neurochemical identities of valop cells and to test for colocalization of the valop isoforms, we used in situ hybridization to characterize the expression of the valop genes along with that of neurotransmitters and a neuropeptide known to be present at the sites of valop expression. Double labeling showed that the thalamic valop population coexpresses valopa and valopb. All the thalamic valop cells overlapped with a GABAergic cell mass that continues from the anterior nucleus to the intercalated thalamic nucleus. A novel valopa cell population found in the superior raphe was serotonergic in nature. A valopb cell population in the Edinger-Westphal nucleus was identified as containing thyrotropin-releasing hormone. Valopb cells localized in the hindbrain intermediate reticular formation were noncholinergic in nature (nonmotorneurons). Thus, the presence of valop cell populations in different brain regions with coexpression of neurotransmitters and neuropeptides and the colocalization of valop isoforms in the thalamic cell population indicate regulatory and functional complexity of VAL-opsin in the brain of the zebrafish.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  17. Lim WL, Soga T, Parhar IS
    Cell Tissue Res, 2014 Feb;355(2):409-23.
    PMID: 24374911 DOI: 10.1007/s00441-013-1765-9
    The migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the preoptic area (POA) from embryonic day 13 is important for successful reproduction during adulthood. Whether maternal glucocorticoid exposure alters GnRH neuronal morphology and number in the offspring is unknown. This study determines the effect of maternal dexamethasone (DEX) exposure on enhanced green fluorescent protein (EGFP) driven by GnRH promoter neurons (TG-GnRH) in transgenic rats dual-labelled with GnRH immunofluorescence (IF-GnRH). The TG-GnRH neurons were examined in intact male and female rats at different postnatal ages, as a marker for GnRH promoter activity. Pregnant females were subcutaneously injected with DEX (0.1 mg/kg) or vehicle daily during gestation days 13-20 to examine the number of GnRH neurons in P0 male offspring. The total number of TG-GnRH neurons and TG-GnRH/IF-GnRH neuronal ratio increased from P0 and P5 stages to P47-52 stages, suggesting temporal regulation of GnRH promoter activity during postnatal development in intact rats. In DEX-treated P0 males, the number of IF-GnRH neurons decreased within the medial septum, organum vasculosom of the lamina terminalis (OVLT) and anterior hypothalamus. The percentage of TG-GnRH neurons with branched dendritic structures decreased in the OVLT of DEX-P0 males. These results suggest that maternal DEX exposure affects the number and dendritic development of early postnatal GnRH neurons in the OVLT/POA, which may lead to altered reproductive functions in adults.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  18. Sahebi M, Hanafi MM, Rafii MY, Azizi P, Abiri R, Kalhori N, et al.
    Biomed Res Int, 2017;2017:9064129.
    PMID: 28191468 DOI: 10.1155/2017/9064129
    Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  19. Volak A, LeRoy SG, Natasan JS, Park DJ, Cheah PS, Maus A, et al.
    J. Neurooncol., 2018 Sep;139(2):293-305.
    PMID: 29767307 DOI: 10.1007/s11060-018-2889-2
    The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  20. Yang C, Li X, Li Q, Zhang B, Li H, Lin J
    Neuroreport, 2017 Dec 06;28(17):1180-1185.
    PMID: 28953094 DOI: 10.1097/WNR.0000000000000903
    Chicken embryos are used widely in the fields of developmental biology and neurobiology. The chicken embryo also serves as a model to analyze gene expression and function using in ovo electroporation. Plasmids may be injected into the spinal cord or tectum of the chicken central nervous system by microinjection for electroporation. Here, we developed a novel method that combines in ovo electroporation and neuronal culturing to study gene function in the chicken tectum during embryo development. Our method can be used to study in-vivo and in-vitro exogenous genes' function. In addition, live cell imaging microscopy, immunostaining, and transfection can be used with our method to study neuronal growth, development, neurite growth and retraction, and axonal pathfinding. Our result showed that axons were present in isolated neurons after culturing for 24 h, and cell debris was low after replacing the media at 48 h. Many GFP-expressing neurons were observed in the cultured cells after 48 h. We successfully cultured the neurons for 3 weeks. Together, this method combines in ovo electroporation and neuronal culturing advantages and is more convenient for the gene function analysis.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
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