Dissertations / Theses on the topic 'EGFP (enhanced green fluorescent protein)'

The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology. Bimolecular fluorescence complementation technique (BiFC)
is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.

The use of fluorescent proteins as fluorescent markers has exploded over the last decades. In particular due to the development of advanced microscopy for live cell measurements, dynamic molecular studies down to single molecule levels and for superresolution microscopy. Many variants of fluorescent proteins exist with varying properties, such as emission color, photostability and brightness. These properties enable advanced applications, like timeresolved imaging or imaging below the diffraction limit. However, the photophysics of fluorescent proteins are complex and in many aspects quite unexplored. The triplet state in particular, is a central photophysical state because it is an entrance gate to an ensamble of deleterious photochemical processes that compromise the photostability of fluorescent proteins.The Pixel team at Institute de Biologie Structurale in France, is mainly focused on developing fluorescent proteins for advanced fluorescence imaging. One of the goals is to understand the influence of photochemistry on the properties of fluorescent proteins.In this project, a method to indirectly observe the triplet state in the prototypical EGFP fluorescent protein was developed. The introduction of new hardware and software, coupled to biophysical experiments, required an interdisciplinary strategy to tackle the obstacles during the route. Experiments under different environmental conditions to test the influence on the population of the triplet state of viscosity, pH, UV and infrared light, triplet state quenchers and temperature were performed.The results show that temperature and laser power greatly influence the triplet state kinetics in EGFP. Notably, it was found that the triplet state lifetime strongly increases at cryotemperature in comparison to roomtemperature. Overall, the newly developed setup and our preliminary results on EGFP open the door to novel studies on the photophysical properties of fluorescent proteins.
Nyttjandet av fluorescerande proteiner som markörer har exploderat de senaste årtionden. Speciellt till följd av utvecklingen av avancerad mikroskopi för levande cellmätningar, dynamiska molekylära studier ned till enstaka molekylnivåer och för superupplösnings mikroskopi. Många varianter av fluorescerande proteiner förekommer med varierande egenskaper så som färg, fotostabilitet och ljusstyrka. Dessa proteiner möjliggör avancerade applikationer, som tidsupplöst bildgivning eller bildgivning med upplösning under diffraktionsgränsen. Fotofysiken bakom fluorescerande proteiner är komplex och i många aspekter ganska outforskad. Triplettillståndet är ett centralt fotofysiskt tillstånd eftersom det är en ingångsport till en rad skadliga fotokemiska processer som äventyrar fotostabiliteten hos fluorescerance proteiner.Pixelteamet på Institute de Biologie Structurale i Frankrike, fokuserar huvudsakligen på utveckling av fluorescerande proteiner för avancerad fluorescerande bildgivning. Ett av målen är att förstå hur fotokemi påverkar egenskaperna hos fluorescerande proteiner.I det här projektet har en metod för att indirekt observera triplettillståndet i det prototypiska fluorescerande proteinet EGFP utvecklats. Introduktionen av ny hårdvara och mjukvara, i kombination med biofysikaliska experiment, krävde en interdisiplinär strategi för att tackla utmaningarna under vägens gång. Experiment under olika miljömässiga förhållanden gjordes för att testa hur populationen av triplettillståndet påverkas till följd av viskositet, pH, UV och infrarött ljus, triplettillståndshämmare och temperatur.Resultaten visar att temperatur och lasereffekt har en stor påverkan på triplettillståndet och dess kinetik hos EGFP. Noterbart är att triplettillståndets livstid ökar kraftigt i kryotemperatur i jämförelse med rumstemperatur. Sammanfattningsvis så utvecklades en ny experimentel uppställning och de tidiga resultaten från EGFP har öppnat dörren för nya studier rörande de fotofysiska egenskaperna hos fluorescerande proteiner.

Orientador: Marcel Otavio Cerri
Resumo: Avanços na biotecnologia proporcionaram possibilidades para o eficiente desempenho da produção em larga escala de diversas biomoléculas e consequentemente suas aplicações industriais. A Escherichia coli se destaca dentre a gama de microrganismos que agem como hospedeiros de genes, desempenhando a função de codificar a síntese proteica. Os vetores mais veiculados na produção de proteínas recombinantes em E. coli são baseados no operon lac, onde o isopropil β-D1-tio-galactopiranosídeo (IPTG), análogo a molécula de lactose, é utilizado para a indução da produção da proteína de interesse. Estudos descritos na literatura também observaram o bom desempenho da lactose como agente de indução da E. coli recombinante na expressão de proteína verde fluorescente melhorada (Enhanced Green Fluorescent Protein – EGFP), e suas vantagens quando comparada ao IPTG, como por exemplo menor custo e menor toxicidade. A EGFP se tornou promissora pelo fato de ser monomérica e não precisar de auxilio de quaisquer agentes adicionais para exibir atividade de fluorescência. Possui variadas utilidades no campo biológico como excelente biomarcador da expressão genica e biosensor. Em bioprocessos, operados em biorreatores convencionais é fundamental o estudo dos parâmetros interferentes nos cultivos para a otimização da expressão do produto desejado. A oxigenação em processos conduzidos de maneira aeróbica, também é uma tarefa desafiadora em biorreatores convencionais, sendo imprescindível o controle da con... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Advances in biotechnology have provided possibilities to the performance of large-scale of biomolecules and therefore of their industrial applications. Escherichia coli stands out among microorganisms that act as host genes, functioning as a synthetic protein. The most useful vectors for the production of recombinant proteins in E. coli are based on the operon lac, where isopropyl β-D1-thiogalactopyroside (IPTG), analogous to lactose, is used to induce the production of proteins of interest. Studies in the literature have also observed the good performance of lactose as an inducer of recombinant E. coli in the expression of Enhanced Green Fluorescent Protein (EGFP), and its advantages when compared to IPTG, such as lower cost and toxicity. EGFP becomes promising because it is monomeric and does not need to help the main agents for fluorescence activity. It has several uses in the biological field as an excellent biomarker of gene expression and biosensor. In bioprocesses, operated in conventional bioreactors, it is fundamental to study the interfering parameters in the cultures to optimize the expression of the desired product. Oxygenation in aerobically conducted processes is also a challenging task in conventional bioreactors, with control of the concentration of dissolved oxygen in the culture medium is essential, which may be limiting for growth and expression of the protein of interest. In processes of production of heterologous proteins, it is assumed that the productiv... (Complete abstract click electronic access below)
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The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal.

The ability of the biological control agents (BCAs) to colonize plant tissues is an important feature involved in microbe-assisted plant protection. Plant-microbe interaction research increased especially in the last decade thanks to technological revolution. Molecular methods and the development of advanced microscopic techniques allow researchers to explore gene expression and localization of beneficial microorganisms within plants. The green fluorescent protein (GFP) and its modified version, enhanced GFP (EGFP), more adapt for expression in mammalian cells and GC-rich actinomycetes like Streptomyces, have been widely used as markers to study gene expression, as well as plant-microbe interactions. Aside fluorescent protein approaches, fluorescence in situ hybridization (FISH) is another frequently used technique to visualize microbial colonization patterns and community composition by application of specific fluorescent probes. Firstly, we transformed five Streptomyces strains, which showed strong inhibition activity against Sclerotinia sclerotiorum, with the EGFP construct by the conjugation method. The conjugation efficiencies varied between the strains, but were comparable to the reference strain. The fitness of transformed strains was similar to wild-type; the transformants maintained similar sporulation, mycelium growth rate, and the ability to produce important secondary metabolites and lytic enzymes. Secondly, two transformed strains, Streptomyces cyaneus ZEA17I, and Streptomyces sp. SW06W, were used to study lettuce colonization dynamics by seed coating method. Their spatio-temporal dynamics were determined in sterile substrate. The strains were consistently recovered from lettuce rhizosphere and inner root tissues up to six weeks. Finally, the colonization pattern of lettuce by Streptomyces cyaneus ZEA17I was examined by both EGFP and FISH approaches combined with confocal laser scanning microscopy (CLSM). For FISH-CLSM analysis, universal bacteria and Streptomyces genus specific probes were used to label S. cyaneus ZEA17I. The consistent presence of the labeled strain at the lettuce root one week after sowing showed that Streptomyces spores could rapidly germinate and produce filamentous mycelium on lettuce. S. cyaneus ZEA17I was detected also on two-week-old roots, indicating the long-term survival ability of this strain in lettuce rhizosphere. Altogether, the antagonistic activity, rhizosphere and root competence showed by the Streptomyces conferred their potential to act as BCA. Further studies on the complex host-pathogen-antagonist interactions will provide additional knowledge to understand the modes and mechanisms of Streptomyces-mediated plant protection.

The Green Fluorescent Protein (GFP) and its numerous variants are applied extensively in a multitude of in vivo applications and have been studied in this context at length. In contrast however, the study of GFP’s within the emerging fields of nanoand micro-technology, which offer broader extracellular applications for GFP and its derivatives, has only recently begun to gather momentum. This thesis presents the directed design of a novel series of Enhanced Green and Enhanced Yellow Fluorescent Proteins (EGFP and EYFP respectively), for implementation in extracellular applications such as biosensing and fundamental research into fluorescence protein behaviour. Each parent fluorescent protein (EGFP or EYFP) was altered to display a single solvent exposed reactive sulfhydryl group with varying degrees of connectivity to the internal GFP chromophore. These sulfhydryl groups were introduced into the protein primary structure via point mutation to yield cysteine residues in place of the targeted native amino acid. Careful examination of the EGFP and EYFP tertiary structures to identify amino acids within the protein primary sequence that fulfilled specific criteria, which were defined in our experimental design, resulted in substitution of amino acids at positions 221, 223, 219, 212 and 97 in EGFP and 221, 223, 212, 95 and 21 in EYFP. Critical development of supporting methodologies delivered vast improvements on literature protocols for expression and purification of the GFP variants listed above. Expression protocol investigation determined that the most prolific E. coli strain for recombinant fluorescent protein production was BL21, which, coupled with our methodology, produced up to 13.6 mg of fluorescent protein per gram of wet cell pellet. The novel purification procedure described in this Thesis delivered highly pure protein with impressive yields (75-80 %). Characterisation of the novel proteins that were designed and produced during this work revealed no change in the proteins’ ability to resist denaturation resulting from cysteine substitution. Neither was there any change in fluorescence emission or UV absorption profiles for standard concentrations (< 60 mM) of any of the purified proteins that were produced. While standard protein solutions returned normal fluorescence emission profiles, solutions that contained protein concentrations above 60 mM displayed red shifted emission maximum values. For protein solutions within the mM concentration range this red shift in fluorescence emission was at times in the order of 30 nm resulting in emission maximums of up to 540 nm for EGFP, and 548 nm for EYFP and recombinant proteins containing an L221C mutation. Preliminary investigations into this phenomenon showed that the changes observed in fluorescence emission were dependent on protein concentration and could be due to dipole-dipole interactions which may be induced by protein aggregate formation at high protein concentrations. Manipulations that were performed on fluorescent proteins during this study included proteolytic digestion with Proteinase K and subsequent testing of the digested protein product. This work identified an increase in the quantum yield of proteolytically digested EGFP and EYFP from 0.6 to 0.8 accompanied by no reduction in the digested proteins resistance to denaturing treatments except when treated with 1 % SDS solution.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

The aim of this project was to investigate the use of the Enhanced Green Fluorescent Protein (EGFP) as a reporter of CFTR promoter activity. six vectors were created coupling portions of the CFTR locus to EGFP in GCVs. Small plasmids were made by conventional cloning procedures, while large PAC vectors were made by a double recombination method employing both homologous and Cre recombinase/loxP recombination. These vectors were transfected into permanent cell lines COS7, MDCK-iowa, T84 and CaCO2, in order to assess the effects of the genomic context elements upon EGFP. The proximal CFTR 5’ region in the p1kbcfproEGFP vector drove expression of the EGFP transgene at low levels in every cell line analysed. This is in agreement with previous reports that show basal levels of CFTR expression driven by this proximal ‘housekeeping’ region. The additional upstream region in the _PAC65bcfproEGFP vector did not appear to modulate expression in any of the cell lines analysed. A comparison of the twin vectors _PACRC1iresEGFP and _PACRC2iresEGFP, which differ only in the absence or presence of CFTR intron 1 respectively, showed similar levels of expression in the COS7 and MDCK-iowa cell lines. Thus, the intron 1 element does not seem to alter expression in these non-gut cell lines; this is consistent with reports that show regulation of CFTR expression in response to the intron element to be specific to cells of the gut epithelium. A comparison of pEGFP-N and _PACRC2cmvEGFP revealed that large PAC vectors show an intrinsic reduction in expression in comparison to their small plasmid counterparts. Further experiments showed that this was not an effect of vector copy number, and that the effect could not act in trans upon a co-transfected molecule. These studies also revealed an unexpected interaction: diluting a reporter plasmid with an anonymous plasmid may actually increase its transfection efficiency. An ex vivo primary air interface sheep tracheal culture was utilised as a more realistic model. Cultures were transfected with several of the genomic context vectors. While PAC vectors had shown a dramatic reduction in expression relative to their small plasmid counterparts in the in vitro studies, only a small reduction was seen to the ex vivo cultures, thus PAC vectors, such as GCVs, may provide a promising approach for gene therapy studies.

Fluorescent proteins (FPs), particularly Enhanced Green Fluorescent Protein (EGFP), are essential tools in the study of intact biological systems. Whilst the photophysics of its progenitor, GFP, have been investigated extensively, far fewer studies of EGFP have been made. In this thesis, a full characterisation of EGFP excited state photophysics by singleand two-photon time-resolved fluorescence lifetime and anisotropy is presented. Furthermore, the two-photon transition tensor, determined by absorption and initial anisotropies, is shown to be dominated by a single element. The two-photon excited state of EGFP was subject to Stimulated Emission Depletion (STED), revealing the stimulated emission cross section, the ground state relaxation time and the time evolution of the higher order distribution moments to which anisotropy is not sensitive. The strong adherence to theoretical Debye diffusion reinforced the conclusions of the two-photon structure model, and showed EGFP to be an excellent molecule for the future development of STED. In addition, these studies provided a sound basis on which to employ single- and two-photon FRET in vivo and in vitro. Cell behaviour is governed by the transduction of molecular signals from the extracellular environment to intracellular compartments. At the centre of the PI 3-kinase signalling pathway is PDK1, a Serine/Threonine kinase, which phosphorylates numerous important downstream targets including Protein Kinase B (PKB). To date however, the regulatory mechanisms governing the behaviour of this protein remain poorly understood. Timeresolved fluorescence lifetime imaging microscopy (FLIM) was employed with FP tagged PDK1 to investigate dynamic interactions in intact cells in situ and in vivo. PDK1 was shown to dimerise in a manner dependent on PI 3-kinase activity and PDK1 PH domain lipid binding. To detail the structure of the observed intermolecular interaction, recombinant FP labelled PDK1 was produced with insect cells. Measurement of the rise in acceptor fluorescence during FRET in vitro indicated the PDK1 dimer pair exists in an antiparallel arrangement. These results provide the first insight on the structure of the dimer and demonstrate that the generation of 3-phosphorylated lipids is required for its formation.

With the introduction of antibiotics in animal feed becoming less popular, the agricultural industry has begun a shift towards the use of probiotics in animal feed. Since there is no current method to evaluate the risks of using genetically modified probiotics in animal feed. The goal of this project was to create a genetically modified model organism for risk assessment. The genetic marker for that was chosen was GFP that was to be expressed on the surface of the cell. The fluorescent properties allow for visualisation of the genetically modified bacteria and the surface expression would allow for the easy capture and recovery of the bacteria for culturing and cell counts. Genome wide screens were performed using the CW PRED algorithm to locate proteins with LPXTG motif for cell wall anchoring. 16 hypothetical proteins were detected and 6 were selected as candidates for possible surface display of GFP. Of these candidates, the novel L. casei protein LSEI_2320 was found to be expressed at the mRNA during early growth by RT PCR and at then protein level during stationary phase with western blot. This LPXTG protein was found at the surface of L. casei ATCC334 during stationary phase and late stationary phase with immunofluorescence microscopy. A genetically modified L. casei ATCC334 was constructed using the surface protein LSEI_2320 locus as a region for recombination with the pRV300 suicide plasmid. Genetic modification of the locus by the insertion of a GFP reporter region just before the predicted signal peptide site resulted in the abrogation of the expression of LSEI_2320 from the cell surface at the late stationary phase. It appears that this particular gene is not necessary to cell survival even though it is abundantly expressed on the cell surface and can be used as a location for genetic modification in L. casei ATCC334.

Fosfolipases D (FLD) do veneno das aranhas do gênero Loxosceles são capazes de causar entre outros efeitos, uma forte agregação plaquetária cujo mecanismo ainda não foi elucidado. Portanto, para estudar o papel das FLDs nesta atividade, uma FLD recombinante de L. gaucho (LgRec1) foi fusionada com a proteína fluorescente verde (EGFP) e utilizada como uma sonda para detectar a interação de LgRec1 com plaquetas. Essa quimera, denominada EGFP-LgRec1, manteve as principais características da LgRec1. A microscopia confocal das plaquetas mostrou que LgRec1 não requer componentes plasmáticos para se ligar às plaquetas, embora estes sejam necessários para que a LgRec1 induza agregação. Além disso, foi observado que a ação da LgRec1 leva à exposição de fosfatidilserina. Contudo, esta exposição não está relacionada à morte celular. Portanto, este trabalho mostrou que uma FLD de Loxosceles se liga a plaquetas, promovendo a exposição de fosfatidilserina, possibilitando a ligação de fatores de coagulação e resultando na agregação plaquetária.
Phospholipases D (PLD) from spider venom of the genus Loxosceles are capable of causing, among other effects, a strong aggregation of platelets and its mechanism has not yet been elucidated. Therefore, to study the role of PLDs in this activity, a recombinant L. gaucho PLD (LgRec1) was fused with a green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 with platelets. This chimera, named EGFP-LgRec1, remained the main activities of LgRec1. Platelet confocal microscopy has shown that LgRec1 does not require plasma components to bind to platelets, although these are required for LgRec1 to induce aggregation. In addition, it has been observed that the action of LgRec1 leads to exposures of phosphatidylserine. However, this exposure is not related to cell death. Therefore, this work showed that a Loxosceles PLD binds to platelets, promoting an exposure of phosphatidylserine, that may act as a scaffold for coagulation factors, resulting in platelet aggregation.

Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens infection. We propose that virus delivery of embryo-inducing genes is a better approach for transformation because A) inserts more than 800 nucleotides are unstable, and will spontaneously inactivate, B) virus DNA can migrate through plasmodesmata to cells around the infected cell, creating a gradient of embryonic potential, C) the virus DNA does not pass through the germ line and the seed will not contain virus. We propose this method of inducing embryogenesis will facilitate the stable transformation of cotton and will be beneficial to the cotton industry. Ectopic expression of AtBBM, AtSTM, and AtWUS GrWUS:meGFP from a constitutive CaMV 35S promoter produced plants with phenotypes similar to those described in previous studies overexpressing AtBBM, indicating that the AtBBM gene was functional. The cotton cotyledon infiltration of the pART27 constructs showed transformed cells in Coker 312 by GFP localization in the nucleus. Although GFP was detected, no visible embryos appeared from the cotyledon. Cotyledons infiltrated with Agrobacterium harboring overexpression vectors withered and aborted after ~2 weeks. The virus-based vector in tissue culture failed to increase transformation efficiency, resulting in no embryos. The combination of hormone concentration showed no contribution to increasing the transformation efficiency.

碩士
國立東華大學
生物技術研究所
97
The Tumor Necrosis Factor (TNF) family constitutes 19 members that mediate diverse biological functions in a variety of cellular systems. The TNF family members regulate cellular functions through binding to membrane-bound receptors belonging to the TNF receptor (TNFR) family. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the Tumor Necrosis Factor (TNF) superfamily. TRAIL and its receptors are the most important role of programmed cell death. TRAIL has been reported to specifically kill various cancer cells but to be not toxic for the majority of normal cells. This was a very favorable point in the application of gene therapy. AID peptides were a new transduction system which was able to deliver biological molecules, for example, protein or DNA. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed a plasmid DNA and transfected it into malignant cells by arginine-rich intracellular peptides (AID peptides). We demonstrated that AID peptides could deliver the plasmid DNA into cancer cells effectively to induce the cancer cells apoptosis phenomenon. In the future, deliver TRAIL with AID peptides may become an effective non-invasive gene therapy.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.

The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model

碩士
國立臺灣大學
藥學研究所
101
Heparin-binding hemagglutinin adhesin (HBHA) is a 28 kDa heparin-binding mycobacterial protein expressed on the surface of Mycobacterium tuberculosis. The C-terminal domain of HBHA dominates adherence to epithelial cells and mediates extra-pulmonary dissemination. The C-terminal region is identified with two repeated motifs : KKAAPA (R1) and KKAAAKK (R2) . In our previous study, one peptide with the sequence of 1R1+2R2 named HBHAc showed the good ability of cell binding, uptake and transport in A549 and Caco-2 cell line. To understandthe the ability and mechanism of HBHAc in protein delivery, enhanced green fluorescence protein (EGFP) fused with HBHAc was used in this study. Fisrt, we used nickel-nitrilotriactic acid (Ni-NTA) to purify histidine tagged EGFP or HBHAc-EGFP overexpressed in E. coli BL21. The human hepatocellular carcinoma cell line, HepG2, was used as an in vitro model. We utilized MTT assay to examine whether the cell viability is affected by recombinant EGFP and HBHAc-EGFP, respectively. We further analyzed the uptake efficiency of HBHAc-EGFP by flow cytometry and confocal microscopy. Immunofluorescence assay was used to investigate the intracellular distribution of HBHAc-EGFP. We also applied chloroquine to investigate the escape of HBHAc-EGFP from endolysosomal pathway. The cellular uptake mechanism of HBHAc-EGFP was studied by studying the effect of different temperature, endocytosis inhibitors, and membrane interaction competitors. Based on SDS-PAGE analysis, EGFP and HBHAc-EGFP recombinant protein was obtained with high purity after purification. No cytotoxicity of both protiens was found in HepG2 cells after 24 h incubation at concentration up to 10 μM. Based on the results of flow cytometry, HBHAc-EGFP efficienctly entered into above 95% of total cells and the mean fluorescence intensity was 25.43 a.u. after 1 h incubation at 1 μM concentration. On the contrary, EGFP only entered into 2% of total cells and the mean fluorescence intensity was 2.97 a.u. under the same condition. Based on the confocal microscopy images, after 1 h incubation at 10 μM concentration, HBHAc-EGFP treated cells showed obvious green fluorescence in the cytoplasm and barely detectable signals were observed in the EGFP treated cells. Furthermore, the transduction of HBHAc-EGFP occurred rapidly and the mean fluorescence intensity gradually intensified until about 1 h of incubation based on the results of flow cytometry and confocal microscopy. However, the mean fluorescence intensity decreased significantly after 2 h incubation. According to the results of immunofluorescence assay, the internalized HBHAc-EGFP was co-localized with early endosome antigen-1 (EEA1) but was not co-localized with caveolin-1. In the endolyosomal blockade assay, HepG2 cells were treated with HBHAc-EGFP and chloroquine for 1 h and replaced with new medium with chloroquine for a period of time. The mean fluorescence intensity measured by flow cyometry remained until 12 h in chloroquine treated cells but dropped dramatically within 1 h in non-chloroquine treated cells. It suggests that chloroquine could effectively block the endolysosomal degradation of HBHAc-EGFP in HepG2 cells. Furthermore, the cellular uptake of HBHAc-EGFP was significantly decreased when cells were incubated at 4℃ or 16℃. In the endocytosis inhibition assay, the entry of HBHAc-EGFP was inhibited by 40% in chlorpromazine treated cells, and methyl-β-cyclodextrin and amiloride only showed about 20% of inhibition. Besides, membrane competitors, heparin and dextran sulfate, greatly reduced the internalization of HBHAc-EGFP up to 90%. These results suggest that the internalization of HBHAc-EGFP fusion protein in HepG2 cells is mostly via clathrin-mediated endocytosis through interaction with heparan sulfate proteoglycans on the plasma membrane. Furthermore, most internalized HBHAc-EGFP was distributed in the endosome and degraded through endolysosmal pathway. However, our previous study showed that HBHAc may utilize direct transduction or lipid-raft endocytosis. In addition, membrane interaction competitors inhibit the cellular uptake of HBHAc-EGFP more effectively than the cellular uptake of HBHAc. HBHAc can effectively deliver EGFP into cells without disrupting the activity of EGFP and no obvious cytototoxicity was observed at the concentrations used for delivering EGFP. Due to the degradation of HBHAc-EGFP by endolysosomal pathway, appropriate endosomal escape strategies are needed for HBHAc to be used as a potiential protein carrier.

碩士
國立成功大學
微生物及免疫學研究所
86
Interleukin 10 (IL-10), expressed mostly in T cells, B cells and macrophages, is a pleiotropic growth and differentiation factor of B cells. It can inhibit the secretion of Th1 cytokines by monocyte, macrophage, and T cells, and exert consequently an important regulatory role in cytokine networks. In order to understand how the expression of IL-10 is regulated, enhanced green fluorescent protein (EGFP) based reporter plasmids under the control of IL-10 promoter sequences were constructed. The DNA fragment spanning from -1212 to -19 region of IL-10 gene was amplified by PCR and confirmed by DNA sequencing. Three IL-10 promtoer clones showing different length in "AC" dinucleotide repeat (32, 36, and 64 bp) were obtained. Two additional clones containing DNA fragment spanning from -746 to -19 or from -558 to -19 were also created. The function of these reporter plasmids had been analyzed in B cell lines: BJAB, and B95/8; T cell lines: Jurkat and Molt-4; and monocyte cell lines: U937 and HL-60. The expression of EGFP was observed directly by fluorescent microscopy and quantitiatively measured by FACScan and fluorescence spectrophotometer. Our results showed that IL-10 promoter was constitutively activated in BJAG, B95/8, and Jurkat but not in Molt-4 and HL-60. DNA transfection caused death of U937 cells. IL-10 reporter activity reduced along with the increasing of the length of the "AC" dinucleotide repeat. The sequences located -1212 to -746 and -746 to -558 showed postitive regulatory activity in BJAB, B95/8, and Jurkat cells. Treatments with phorbol myristate acetate (PMA), calcium ionophore (A23187), or PMA combined A23187 had no significant effects on the IL-10 reporter expression in Jurkat and Molt-4 cells. Lipopolysaccharide (LPS) had no effect on BJAB, B95-8, and HL-60. The expression of EGFP in the tested cell lines was not effected by dehydroepiandrosterone. Although the PMA or LPS could not augment the reporter activities in our tested cell lines, it increased the IL-10 mRNA expression in Jurkat and U937 cells as detected by RT-PCT. These results suggested that there were still some other regulatory elements on IL-10 promoter upstream of -1212 or downstream -of 19.

碩士
國立成功大學
微生物暨免疫學研究所
87
IL-10 is an important immunosuppressive and anti-inflammatory cytokine. It is mainly produced by T cells, B cells and macrophages in vivo. It can inactivate macrophages and dendritic cells to loss the ability in T and NK cells activation due to inhibition of cytokines synthesis, MHC class II presentation, and costimulator expression. In constract, it promotes B cells and mast cells differentiation and proliferation. Besides, it involves in the tumorgenesis of comman tumors and in the pathogenesis of autoimmune diseases. To understand how it is regulated, especially on IL-10 promoter, we constructed reporter plasmids using the enhanced green fluorescent protein (EGFP) under the control of IL-10 promoter sequences spanning from -1212 to -19 (pEGFP10p), from -746 to -19(pEGFPd3), and from -558 to -19 (pEGFPd) regions. We established Jurkat and BJAB cell lines stably containing EGFP-reporter plasmids under the selection by G418. EGFP expressions driven by the three different fragments of IL-10 promoter were analyzed in the stable cells upon treatments with mitogens, forskolin, cyclosporine A, dexamethasone, staurosporine, and active Ras overexpression. Besides, we also analyzed dynamic EGFP-expression in stable cells when cocultured with Jurkat, BJAB, U-937 cells, hepatoma cells, glioma cells, and peripheral mononuclear cells and plasma of healthy people or SLE patients. We found that the IL-10 promtoer activities were differentially regulated in BJAB and Jurkat cells upon mitogen and chemical agents. In coculture experiments, activated Jurkat, BJAB and U-937 enhanced the activities of IL-10 promoter in BJAB stable cells but not that in Jurkat stable cells. Glioma cells enhanced the IL-10 promoter activity of pEGFP10p in Jurkat stable cells and suppressed that in BJAB stable cells. Hepatoma cells suppressed the IL-10 promoter activity of pEGFP10p but not that of pEGFPd3 and pEGFPd in BJAB stable cells. PBMC and plasma of SLE patients had various effects on IL-10 promoter activity. In conclusion, the activities of IL-10 promoter in Jurkat and BAJB cells were differently regulated. The stable cells with EGFP reporter plasmids may serve as a convenient biosensor system to study the gene regulation.

碩士
明志科技大學
生化工程研究所
96
The method development for direct capture of His-tag enhanced green fluorescent protein (His-EGFP) from unclarified E.coli homogenates using stirred fluidized bed (SFB) has been described. Cu(II), Zn(II), Ni(II) and Co(II) metal ion were evaluated in immobilized metal affinity chromatography (IMAC) for the recovery of His-EGFP. Immobilized Ni(II) chelating adsorbent was found to be the most effective for capture of His-EGFP. The adsorption characteristic of Ni(II)-STREAMLINE chelating adsorbent used in this work for total protein and His-EGFP in unclarified E.coli homogenates has been assessed by the measurements of equilibrium adsorption isotherm and uptake rate. Optimal conditions for the elution of His-EGFP were also investigated in packed bed experiments conducted with clarified E.coli homogenates. On the basis of the results, direct capture of His-EGFP from unclarified E.coli homogenates by SFB was carried out under the different loading volumes (50-200 ml). The purification results showed that the His-EGFP was directly recovered using 150 mM Imidazol buffer (pH 7) from unclarified E.coli homogenate with a purification factor of 2.04 and yield of 73.6% in the single step. Other contaminants were completely eluted by using 25 mM EDTA ( pH 7).

碩士
明志科技大學
生化工程研究所
102
In this work, polyacrylonitrile nanofiber membrane (PAN) was prepared by using electrostatic spinning technique. After a series of treatment for the PAN membrane, the modified membrane was finally functionalized with bromoacetic acidic (BrA), and iminodiacetic acidic (IDA) groups, respectively. The modified membranes wre chelated by Cu(II), Co(II), Ni(II), or Zn(II) as immobilized metal affinity nanofibrous (IMAN) membrane (abbrev. BrA-M or IDA-M). Recombinant E. coli cells containing enhanced green fluorescent protein (EGFP) was disrupted by ultrasonic technique. The isotherm and kinetic adsorption experiments were carried out by using clarified feedstock in a bath stirred tank system. The isotherm data for general protein and EGFP were well correlated by the Freundlich model for both IMAN membranes chelated by these metal ions. In kinetic experiments, the kinetic data for IMAN membrane were tested by using pseudo-first-order reaction and pseudo-second-order models. Generally speaking, the kinetic studies for general protein and EGFP showed that the adsorption followed a pseudo-second-order reaction. Its rate constant k2 increased with increasing concentrations of adsorption, indicating that the increased concentration of E. coli sample accelerated the adsorption rate. In membrane adsorption chromatography for both IMAN membranes, the dynamic binding capacity (DBC) was evaluated by adsorption breakthrough curve and calculated under different chelated metal ions and flow rates, respectively. The DBC for IMAN membrane was found to be lower at higher operating flow rate. For IDA-M adsorption process at a flow rate of 0.1 ml/min, the order of DBC for the general protein was Co(II) (40.0 mg/g)= Zn(II) (40.0 mg/g )> Cu(II) (16.2 mg/g)> Ni(II) (16.2 mg/g); the order of DBC for EGFP was Co(II) (5.77×106 AU/g)> Zn(II) (4.77×106 AU/g)> Cu(II) (3.47×106 AU/g)> Ni(II) (2.73×106 AU/g). However, for BrA-M adsorption process at the same flow rate, the order of DBC for the general protein was Ni(II) (31.6 mg/g)> Cu(II) (27.4 mg/g )> Zn(II) (21.4 mg/g)> Co(II) (10.6 mg/g); the order of DBC for EGFP was Ni(II) (8.25×106 AU/g)> Cu(II) (4.90×106 AU/g)> Co(II) (3.60×106 AU/g)> Zn(II) (3.41×106 AU/g). Based on the adsorption breakthrough curves, the results showed that IDA-M or BrA-M as IMAN membrane was not suitable for use in the purification of EGFP by using membrane chromatography.

碩士
明志科技大學
生化工程研究所
100
In this study, the optimal conditions for the production of recombinant enhanced green fluorescent protein (His-EGFP) expressed in Escherivhia coli BL21 (DE3) grown in LB media were investigated in a shaker flask. Firstly, five key parameters (e.g. induction concentration, induction time, culture pH, culture time, and induction temperature) were investigated by single factor tests to find the operating range for applying to full factorial design (FFD). The results indicated that induction time and culture time have a significant effect on EGFP production. Secondly, response surface methodlogy (RSM) based on central composite design (CCD) was applied to obtain the optimal conditions for the production of EGFP. The optimal conditions was found to be induction concentration of 1 mM, induction time of 12 hr, culture pH of 5,culture time of 20 hr, and culture temperature of 20℃. Under optimal conditions, the model predicted EGFP intensity was 1.278×105 U/ml which is equal to the average experimental value of 1.275×105 U/ml. The obtained optimal environmental conditions were then employed to scale up for EGFP production in a 5 L bioreactor.

碩士
明志科技大學
生化工程研究所
97
In this study, experimental design was used to optimize the environmental factors for the production of EGFP (enhanced green fluorescent protein). A 2-level-3-factor fractional factorial design (FFD) was employed to evaluate the effects of operating parameters on EGFP production at a shaker rate of 230 rpm, such as initial culture pH, induction time, and induction temperature. The individual and interactive effects of the three independent variables on the production of EGFP were studied and the optimal EGFP production was found to be at initial pH of 7.0, induction time at 1.75 h after cultivation (OD600 approach to 0.7–0.9), and induction temperature of 26℃. The obtained optimal environmental conditions were then employed to scale up for EGFP production in a 5 L bioreactor. Subsequently, the collected cells were suspended with buffer and disrupted by high pressure homogenizer for three cycles at 20 Kpsi and 4℃. The release of EGFP and total protein from 50% (w/v) disrupted cells was 7.05×104 AU/ml and 36.86 mg/ml, respectively. The ion exchange adsorbent (i.e. STREAMLINE DEAE) was further used to evaluate the adsorption efficiency for EGFP from highly unclarified E. coli feedstock (50% w/v). The effects of rotating speed (0-200 rpm) and feedstock velocity (100-300 cm/h) on the adsorption efficiency were investigated by using stirred fluidized bed chromatography. The results showed that the optimal operating conditions were found to be rotating speed of 100 rpm and feedstock velocity of 100 cm/h. The recovery yield of EGFP was up to 96.3% with a purification factor of 2.9.

The protein transduction domain (PTD) of the HIV-I TAT protein has been shown to be capable of crossing cellular membranes and even the blood-brain barrier while carrying cargo molecules along with it. Exploiting this property to deliver biologically active acid β-glucosidase (GBA) would be of use to improve the current treatment of Gaucher disease by enzyme replacement therapy. Genetic fusion of the TAT PTD to GBA was performed and the resulting gene was inserted into an insect expression vector, p2ZOptcxF, to allow for heterologous protein production in Sf9 cells. A TAT fusion with enhanced green fluorescent protein (EGFP) was also created to serve as a control. The insect vector encoded a cellulose-binding domain to allow for affinity purification of the heterologous proteins. The Sf9 system produced 0.9-1.2 μg/ml quantities of EGFP fusion proteins, but only low ng/ml levels of GBA fusions. The addition of the cellulose binding domain decreased protein expression, but could be used for purification when μg/ml quantities of protein were produced. This suggests that expression of soluble, unglycosylated proteins such as EGFP can be achieved in the p2ZOptcxF/Sf9 system and to a lesser extent complex, highly-glycosylated proteins such as GBA can also be produced. In order to improve the expression of GBA fusions further optimization of the vector, selection and production must be undertaken.

碩士
明志科技大學
生化工程研究所
96
Statistics based experimental designs were used to optimize the medium components and environmental factors for EGFP production. Preliminary studies on the factors enabled identification of night variables important with respect to EGFP production. The most important factors with respect to E. coli BL21 was then identified using the 2-level fractional factorial design. In general, glucose, tryptone, peptone, NaCl, Na2HPO4, KH2PO4, MgSO4•7H2O, CaCl2•2H2O and trace elements affected EGFP production. CCD was used for model building and glucose and FeSO4 were found to have an effect on EGFP production in E. coli. The optimal values of important variables were determined by response surface methodology (RSM). The optimal conditions for the production of EGFP were as follows 4.215 g/L glucose，2.750 g/L tryptone，1.375 g/L peptone，4.750 g/L NaCl，6 g/L Na2HPO4，10.5 g/L KH2PO4， 1.01 g/L MgSO4•7H2O，0.039 g/L CaCl2•2H2O，0.297 mg/L FeSO4，1 mg/L CuSO4，1 mg/L NiSO4. This optimization strategy led to an increase in EGFP production in the E. coli by 333.1% as compared modified M9 medium developed by Lu (2007).

碩士
明志科技大學
生化工程研究所
95
An improved procedure was developed involving a stirred fluidized bed (SFB) adsorption for direct recovery of recombinant enhanced green fluorescent protein (EGFP) from unclarified E. coli homogenates. The maximal binding capacity of STREAMLINE DEAE adsorbent for EGFP was observed at pH 9. A typical adsorption isotherm curve for EGFP in a crude E. coli homogenate was well correlated with the Langmuir model. The maximal binding capacity for EGFP and dissociation constant for EGFP-adsorbent complex were 6.3mg/ ml and 1.3x10-3 mg/ ml respectively. Optimal conditions for elution scheme were further determined in small packed bed experiments conducted with clarified E.coli homogenate under the various operating conditions. The recovery yield for the EGFP was approximately 93% by using 0.2 M NaCl (pH 9) and 10 fold adsorbent volumes at liquid velocity of 200 cm/h. The operating conditions were chosen for use in further purification process. Finally, the experiments were carried out to assess the feasibility of using SFB chromatography for direct recovery of EGFP from different loading volumes (50-200 ml) of highly crude E.coli homogenate (pH 9). It was found to be successful in achieving purification of EGFP in a high yield of 95-98% and the purification factor was approx. 3 in a single step under these loading volumes of E. coli homogenates at a rotating speed of 200 rpm.