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Sample records for fluorescent protein transgenic

  1. Generation of red fluorescent protein transgenic dogs.

    Science.gov (United States)

    Hong, So Gun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Teoan; Kwon, Mo Sun; Koo, Bon Chul; Ra, Jeong Chan; Kim, Dae Yong; Ko, CheMyong; Lee, Byeong Chun

    2009-05-01

    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP.

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

    Science.gov (United States)

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

    2011-06-01

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

  3. First molecular identification of the transgene red fluorescent protein (RFP) in transgenic ornamental zebrafish (Danio rerio) introduced in Peru

    OpenAIRE

    Carlos Scotto; Fernando Serna

    2013-01-01

    In this paper the transgenic fluorescent red, orange and pink zebra fish (Danio rerio), found in local aquariums in Peru, were identified using the PCR technique to amplify the transgene RFP sea anemone belonging to Discosoma spp. The gene expression of the red fluorescent protein (RFP) transgene was found to determine different gradients-of-bioluminescence (shades in color) in each GMO fish analyzed. We performed sequence analysis of the two variants of the RFP along with six variants of the...

  4. Transgenic expression of green fluorescent protein in mouse oxytocin neurones.

    Science.gov (United States)

    Young, W S; Iacangelo, A; Luo, X Z; King, C; Duncan, K; Ginns, E I

    1999-12-01

    Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in-frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell-specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.

  5. First molecular identification of the transgene red fluorescent protein (RFP in transgenic ornamental zebrafish (Danio rerio introduced in Peru

    Directory of Open Access Journals (Sweden)

    Carlos Scotto

    2013-09-01

    Full Text Available In this paper the transgenic fluorescent red, orange and pink zebra fish (Danio rerio, found in local aquariums in Peru, were identified using the PCR technique to amplify the transgene RFP sea anemone belonging to Discosoma spp. The gene expression of the red fluorescent protein (RFP transgene was found to determine different gradients-of-bioluminescence (shades in color in each GMO fish analyzed. We performed sequence analysis of the two variants of the RFP along with six variants of the existing fluorescent protein GFP from the Genbank, this could help identify quickly if they are new genes or variants thereof as these novel fluorescent proteins may be introduced in aquatic GMO in the future. Thus, developing and improving biosecurity measures through its timely detection at the molecular genetic level.

  6. The Cyan Fluorescent Protein (CFP) Transgenic Mouse as a Model for Imaging Pancreatic Exocrine Cells

    Science.gov (United States)

    Cao, Hop S Tran; Kimura, Hiroaki; Kaushal, Sharmeela; Snyder, Cynthia S; Reynoso, Jose; Hoffman, Robert M; Bouvet, Michael

    2015-01-01

    Context The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. Objective To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. Design Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA). Intervention All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. Main outcome measure Determination of fluorescence intensity of different organs. Results Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. Conclusions The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer. PMID:19287108

  7. The Cyan Fluorescent Protein (CFP Transgenic Mouse as a Model for Imaging Pancreatic Exocrine Cells

    Directory of Open Access Journals (Sweden)

    Hop S Tran Cao

    2009-03-01

    Full Text Available The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. Objective To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. Design Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan. Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA. Intervention All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. Main outcome measure Determination of fluorescence intensity of different organs. Results Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. Conclusions The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.

  8. Post-mortem re-cloning of a transgenic red fluorescent protein dog

    OpenAIRE

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

    2011-01-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Althoug...

  9. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    Science.gov (United States)

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

    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.

  11. Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

    DEFF Research Database (Denmark)

    Huber, Reinhard C.; Remuge, Liliana; Carlisle, Ailsa

    2012-01-01

    Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology—animal welfare—has not been approached through systematic assessment...... and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals...... months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs...

  12. Quantitative analysis of tetracycline-inducible expression of the green fluorescent protein gene in transgenic chickens.

    Science.gov (United States)

    Koo, Bon Chul; Kwon, Mo Sun; Roh, Ji Yeol; Kim, Minjee; Kim, Jin-Hoi; Kim, Teoan

    2012-01-01

    The use of transgenic farm animals as "bioreactors" to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side-effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognized limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet. As a proof of principle study, however, quantitative assessment of expression was not possible, as only one G0 and one G1 transgenic chicken was obtained. In the current study, a sufficient number of G2 and G3 transgenic chickens were obtained, and quantification analysis demonstrated up to a 20-fold induction of expression by doxycycline. In addition, stable transmission of the transgene without any apparent genetic modifications was observed through several generations. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene. Importantly, these results also support the use of the retroviral system for generating transgenic animals with minimal risk in terms of biosafety.

  13. Post-mortem re-cloning of a transgenic red fluorescent protein dog.

    Science.gov (United States)

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

    2011-12-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

  14. Dexamethasone-Inducible Green Fluorescent Protein Gene Expression in Transgenic Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Wei Tang; Hilary Collver; Katherine Kinken

    2004-01-01

    Genomic research has made a large number of sequences of novel genes or expressed sequence tags available. To investigate functions of these genes, a system for conditional control of gene expression would be a useful tool. Inducible transgene expression that uses green fluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines of cotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir (NOR; Abies nordmanniana Lk.), and rice (RIC; Oryza sativa L. Cv. Radon). Transgenic cell lines were used to test the function of the chemical inducer dexamethasone. Inducible transgene expression was observed with fluorescence and confocal microscopy, and was confirmed by northern blot analyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h after treatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgenic cells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducer for optimum inducible gene expression system varied among transgenic cell lines. The inducible gene expression system described here was very effective and could be valuable in evaluating the function of novel gene.

  15. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

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    Mazahir T Hasan

    2004-06-01

    Full Text Available Genetically encoded fluorescent calcium indicator proteins (FCIPs are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs and synaptic and sensory stimulation can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

  16. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

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    Stefanie Besser

    Full Text Available GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65. TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.

  17. Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

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    Mann David GJ

    2012-05-01

    Full Text Available Abstract Background The expression of fluorescent protein (FP genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

  18. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

    Science.gov (United States)

    Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

  19. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    Science.gov (United States)

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  20. Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

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    Inagaki, Yoshinori; Matsumoto, Yasuhiko; Ishii, Masaki; Uchino, Keiro; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2015-06-10

    In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.

  1. New Wistar Kyoto and spontaneously hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein

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    Ana Isabel Garcia Diaz

    2016-04-01

    Full Text Available The Wistar Kyoto (WKY rat and the spontaneously hypertensive (SHR rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN and metabolic syndrome, respectively. Novel transgenic (Tg strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP under the rat elongation factor 1 alpha (EF1a promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.

  2. Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector.

    Science.gov (United States)

    Koo, Bon Chul; Kwon, Mo Sun; Choi, Bok Ryul; Kim, Jin-Hoi; Cho, Seong-Keun; Sohn, Sea Hwan; Cho, Eun Jung; Lee, Hoon Taek; Chang, Wonkyung; Jeon, Iksoo; Park, Jin-Ki; Park, Jae Bok; Kim, Teoan

    2006-11-01

    The Moloney murine leukemia virus (MoMLV) -based retrovirus vector system has been used most often in gene transfer work, but has been known to cause silencing of the imported gene in transgenic animals. In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of enhanced green fluorescent protein (eGFP). The level of eGFP expression was conserved after germline transmission and as much as 100 microg of eGFP could be detected per 1 mg of tissue protein. DNA sequencing showed that the transgene had been integrated at chromosome 26 of the G1 and G2 generation transgenic chickens. Owing to the stable integration of the transgene, it is now feasible to produce G3 generation of homozygous eGFP transgenic chickens that will provide 100% transgenic eggs. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors.

  3. Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LIU ZhongHua; SUN Shuang; LI YuTian; WANG HongBin; R S PRATHER; SONG Jun; WANG ZhenKun; TIAN JiangTian; KONG QingRan; ZHENG Zhong; YIN Zhi; GAO Li; MA HaiKun

    2008-01-01

    Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 plJmL liposome and 1.6 pg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

  4. 204 POTENTIAL OF GREEN FLUORESCENT PROTEIN LOCUS FOR GENE EDITING IN DNA TRANSPOSON-PRODUCED TRANSGENIC CATTLE.

    Science.gov (United States)

    Yum, S-Y; Lee, S-J; Kim, H-M; Lee, C-I; Kim, H-S; Kim, H-J; Choi, W-J; Hahn, S-E; Lee, J-H; Kim, S-J; Jang, G

    2016-01-01

    Recently, we published on the efficient production of transgenic cattle using the DNA transposon system (Yum et al. 2016 Sci. Rep. 6, 27185). In that study, 8 transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon) via microinjection of zygotes. In the analysis of their genomic stability using next-generation sequencing, there was no significant difference in the number of genomic variants between transgenic and nontransgenic cattle. In this study, we have described current status of those transgenic cattle in term of health, germ-line transmission, and application. All the transgenic cattle have grown up to date (the oldest being 30 months old, the youngest being 12 months old) without any health issue. In general blood analysis, there were not any significant changes between transgenic cattle and wild type. Because the transgene (green fluorescent protein; GFP) expression is constitutively active and has strong expression, it could be visualised without fluorescence equipment. One of transgenic male cattle reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for IVF. In every IVF replication, around 80% blastocysts expressed the GFP. Over 36 GFP blastocysts were frozen for embryo transfer in the future, and we are planning to crossbreed for generating homozygotic transgenic cattle. Another application is to use the GFP locus to gene-edit the transgenic cattle, as long-term expression of transgene did not affect their health. In 1 cell stage, embryos produced using GFP frozen-thawed semen, single guide RNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of single guide RNA target site, were co-injected and RFP was observed. Knockout/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we

  5. Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein.

    Science.gov (United States)

    Ge, Jiachun; Dong, Zhangji; Li, Jingyun; Xu, Zhiqiang; Song, Wei; Bao, Jie; Liang, Dong; Li, Junbo; Li, Kui; Jia, Wenshuang; Zhao, Muzi; Cai, Yongxiang; Yang, Jiaxin; Pan, Jianlin; Zhao, Qingshun

    2012-10-01

    Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of β-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

  6. Transfection of Eimeria mitis with yellow fluorescent protein as reporter and the endogenous development of the transgenic parasite.

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    Mei Qin

    Full Text Available BACKGROUND: Advancements have been made in the genetic manipulation of apicomplexan parasites. Both the in vitro transient and in vivo stable transfection of Eimeria tenella have been developed successfully. Herein, we report the transient and stable transfection of Eimeria mitis. METHODS AND FINDINGS: Sporozoites of E. mitis transfected with enhanced yellow fluorescent protein (EYFP expression plasmid were inoculated into chickens via the cloacal route. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting (FACS and then passaged 6 generations successively in chickens. The resulting population was analyzed by genome walking and Western blot. The endogenous development of the transgenic E. mitis was observed and its reproduction potential was tested. The stable transfection of E. mitis was developed. Genome walking confirmed the random integration of plasmid DNA into the genome; while Western blot analysis demonstrated the expression of foreign proteins. Constitutive expression of EYFP was observed in all stages of merogony, gametogony and sporogony. The peak of the transgenic oocyst output was delayed by 24 h and the total oocyst reproduction was reduced by 7-fold when compared to the parental strain. CONCLUSION: Stable transfection of E. mitis was successfully developed. The expression of foreign antigens in the transgenic parasites will facilitate the development of transgenic E. mitis as a vaccine vector.

  7. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    Science.gov (United States)

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

    2009-09-01

    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

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

    Science.gov (United States)

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

    2014-05-01

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

  9. Kisspeptin regulates gonadotropin-releasing hormone secretion in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats

    Institute of Scientific and Technical Information of China (English)

    Haogang Xue; Chunying Yang; Xiaodong Ge; Weiqi Sun; Chun Li; Mingyu Qi

    2013-01-01

    Kisspeptin is essential for activation of the hypothalamo-pituitary-gonadal axis. In this study, we established gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. Rats were injected with 1, 10, or 100 pM kisspeptin-10, a peptide derived from full-length kisspeptin, into the arcuate nucleus and medial preoptic area, and with the kisspeptin antagonist peptide 234 into the lateral cerebral ventricle. The results of immunohistochemical staining revealed that pulsatile luteinizing hormone secretion was suppressed after injection of antagonist peptide 234 into the lateral cerebral ventricle, and a significant increase in luteinizing hormone level was observed after kisspeptin-10 injection into the arcuate nucleus and medial preoptic area. The results of an enzyme-linked immunosorbent assay showed that luteinizing hormone levels during the first hour of kisspeptin-10 infusion into the arcuate nucleus were significantly greater in the 100 pM kisspeptin-10 group than in the 10 pM kisspeptin-10 group. These findings indicate that kisspeptin directly promotes gonadotropin-releasing hormone secretion and luteinizing hormone release in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. The arcuate nucleus is a key component of the kisspeptin-G protein-coupled receptor 54 signaling pathway underlying regulating luteinizing hormone pulse secretion.

  10. Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism.

    Science.gov (United States)

    Fujiki, Yutaka; Tao, Kai; Bianchi, Diana W; Giel-Moloney, Maryann; Leiter, Andrew B; Johnson, Kirby L

    2008-02-01

    Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P = 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both

  11. Improving the production of transgenic fish germlines: in vivo evaluation of mosaicism in zebrafish (Danio rerio using a green fluorescent protein (GFP and growth hormone cDNA transgene co-injection strategy

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    Márcio de Azevedo Figueiredo

    2007-01-01

    Full Text Available In fish, microinjection is the method most frequently used for gene transfer. However, due to delayed transgene integration this technique almost invariably produces mosaic individuals and if the gene is not integrated into germ cells its transmission to descendants is difficult or impossible. We evaluated the degree of in vivo mosaicism using a strategy where a reporter transgene is co-injected with a transgene of interest so that potential germline founders can be easily identified. Transgenic zebrafish (Danio rerio were produced using two transgenes, both comprised of the carp beta-actin promoter driving the expression of either the green fluorescent protein (GFP reporter gene or the growth hormone cDNA from the marine silverside fish Odonthestes argentinensis. The methodology applied allowed a rapid identification of G0 transgenic fish and also detected which fish were transmitting transgenes to the next generation. This strategy also allowed inferences to be made about genomic transgene integration events in the six lineages produced and allowed the identification of one lineage transmitting both transgenes linked on the same chromosome. These results represent a significant advance in the reduction of the effort invested in producing a stable genetically modified fish lineage.

  12. Glyphosate inhibits the translocation of green fluorescent protein and sucrose from a transgenic tobacco host to Cuscuta campestris Yunk.

    Science.gov (United States)

    Nadler-Hassar, Talia; Goldshmidt, Alexander; Rubin, Baruch; Wolf, Shmuel

    2004-09-01

    The parasitic plant Cuscuta campestris is dependent on its host for water, assimilates and amino acids. It can be controlled by the herbicide glyphosate, which inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), resulting in shikimate accumulation. In this study, C. campestris was parasitic on transgenic tobacco plants expressing green fluorescent protein (GFP) in the phloem. Changes in [14C]sucrose and GFP accumulation in the parasite were used as indicators of the herbicide's effect on translocation between the host and parasite. Host plants were treated with glyphosate 22 days after sowing. Shikimate accumulation in the parasite 1 day after glyphosate treatment (DAGT) confirmed EPSPS inhibition in C. campestris. No damage was visible in the host plants for the first 3 DAGT, while during that same time, a significant reduction in [14C]sucrose and GFP accumulation was observed in the parasite. Thus, we propose that the parallel reduction in GFP and sucrose accumulation in C. campestris is a result of a glyphosate effect on the parasite's ability to withdraw assimilates from the host.

  13. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    BOU; ShorGan

    2009-01-01

    In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  14. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GUO XuDong; YANG DongShan; Ao XuDong; WU Xia; LI GuangPeng; WANG LingLing; BAO MingTao; XUE Lian; BOU ShorGan

    2009-01-01

    In the present study, cashmere goat fetal flbroblasta were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1(IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasta cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h.Parthenogenetic ooctyes were used as a model to Investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% va 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05).After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysta were randomly selected to identify transgene by polymeraee chain reaction. Both were positive. These results showed that: (i) RFP and Neo genes were correctly expressed indicating that transgenlc somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  15. Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Yunhai; PAN; Dengke; SUN; Xiuzhu; SUN; Guojie; WANG; Xiaobo; LIU; Xiaohui; LI; Yan; DAI; Yunping; LI; Ning

    2006-01-01

    In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O2) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O2) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.3%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MⅡ oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.

  16. Characterization of sensory neuron subpopulations selectively expressing green fluorescent protein in phosphodiesterase 1C BAC transgenic mice

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    Anderson Rebecca L

    2006-05-01

    Full Text Available Abstract Background The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c BAC transgenic mice in which a green fluorescent protein (GFP reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord. Results Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative. A small proportion of both non-peptidergic (IB4-binding and peptidergic (CGRP immunoreactive subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X3. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn. Conclusion The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.

  17. Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research.

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    Laura H Comley

    Full Text Available BACKGROUND: Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that thy1-driven expression of yellow fluorescent protein (YFP in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury. CONCLUSIONS/SIGNIFICANCE: We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.

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

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    Wang Yi

    2011-11-01

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

  19. Mapping of fluorescent protein-expressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice.

    Science.gov (United States)

    Porrero, Cesar; Rubio-Garrido, Pablo; Avendaño, Carlos; Clascá, Francisco

    2010-07-23

    Transgenic mouse lines in which a fluorescent protein is constitutively expressed under the Thy1 gene promoter have become important models in cell biology and pathology studies of specific neuronal populations. As a result of positional insertion and/or copy number effects on the transgene, the populations expressing the fluorescent protein (eYFP+) vary markedly among the different mice lines. However, identification of the eYFP+ subpopulations has remained sketchy and fragmentary even for the most widely used lines such as Thy1-eYFP-H mice (Feng, G., Mellor, R.H., Bernstein, M., Keller-Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman and J.W., Sanes. J.R. 2000. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41-51). Here, we provide a comprehensive mapping of labeled cell types throughout the central nervous system in adult and postnatal (P0-P30) Thy1-eYFP-H mice. Cell type identification was based on somatodendritic morphology, axon trajectories, and, for cortical cells, retrograde labeling with Fast Blue to distinguish between subpopulations with different axonal targets. In the neocortex, eYFP+ cells are layers 5 and 6 pyramidal neurons, whose abundance and sublaminar distribution varies markedly between areas. Labeling is particularly prevalent in the corticospinal cells; as a result, the pyramidal pathway axons are conspicuously labeled down to the spinal cord. Large populations of hippocampal, subicular and amygdaloid projection neurons are eYFP+ as well. Additional eYFP+ cell groups are located in specific brainstem nuclei. Present results provide a comprehensive reference dataset for adult and developmental studies using the Thy1-eYFP-H mice strain, and show that this animal model may be particularly suitable for studies on the cell biology of corticospinal neurons.

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

  1. The developmental expression of fluorescent proteins in organotypic hippocampal slice cultures from transgenic mice and its use in the determination of excitotoxic neurodegeneration

    DEFF Research Database (Denmark)

    Noraberg, Jens; Jensen, Carsten V; Bonde, Christian

    2007-01-01

    Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death...... in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological...... subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four...

  2. Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

    2016-06-22

    In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

  3. Electrophysiological effects of kainic acid on vasopressin-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 neurones isolated from the supraoptic nucleus in transgenic rats.

    Science.gov (United States)

    Ohkubo, J; Ohbuchi, T; Yoshimura, M; Maruyama, T; Ishikura, T; Matsuura, T; Suzuki, H; Ueta, Y

    2014-01-01

    The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)-producing and oxytocin (OXT)-producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP-enhanced green fluorescent protein (eGFP) and the other expressing an OXT-monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP-eGFP and OXT-mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate-induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP-eGFP and OXT-mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA-induced responses were inhibited by the non-NMDA ionotrophic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3(1H4H)-dione in both AVP-eGFP and OXT-mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose-dependent manner. The KA-induced currents were significantly larger in OXT-mRFP1 neurones than in AVP-eGFP neurones. This significant difference in KA-induced currents was abolished by the GluK1-containing KAR antagonist UBP302. At high concentrations (250-500 μm), the specific GluK1-containing KAR agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) induced significantly larger currents in OXT-mRFP1 neurones than in AVP-eGFP neurones. Furthermore, the difference between the AVP-eGFP and OXT-mRFP1 neurones in the ATPA currents

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

    Directory of Open Access Journals (Sweden)

    Kofuji Takefumi

    2011-05-01

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

  5. Transgenic expression of green fluorescent protein in caprine embryos produced through electroporation-aided sperm-mediated gene transfer.

    Science.gov (United States)

    Kumar Pramod, R; Kumar, Rakesh; Mitra, Abhijit

    2016-01-15

    Current methods of transgenic animal production are afflicted by low efficiency and high cost. Recently, the electroporation aided sperm-mediated gene transfer (SMGT) emerges as a promising alternative with variable success rate. Among the domestic animal species, the electroporation-aided SMGT is less investigated in goats, except a few reports in which attempts have been made using the auto-uptake method of SMGT. In this study, we report an optimized electroporation condition for SMGT of caprine sperm cells. Results of this study demonstrated that electroporation of caprine sperm cells at 300 V for 200 mS in TALP medium allowed the maximum uptake of foreign DNA with minimum adverse effects on the vital semen parameters viz., progressive motility, viability, and membrane and acrosome integrity. Further, DNA binding assay revealed DNA uptake by 81.3% sperm cells when 1.0 μg of DNA was used under optimum electroporation conditions as compared to 16.5% on simple incubation. The qPCR analysis showed four-fold more (Pelectroporation than incubation. A similar cleavage rate was observed after IVF using either electroporated (23.20 ± 1.20) or non-electroporated (25.20 ± 2.41) sperm cells suggesting the absence of adverse effect of electroporation on the fertilizing ability. Out of the 116 embryos produced by electroporated sperm, five (4.31%) embryos showed the expression of the foreign gene. In conclusion, our results confirm that using optimized electroporation conditions, the caprine sperm cells can uptake foreign DNA effectively with minimum negative effect on the semen parameters and could produce transgenic embryos.

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

    OpenAIRE

    2011-01-01

    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 prot...

  7. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  8. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  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.

    Science.gov (United States)

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

    2009-05-01

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

  10. 红色荧光蛋白基因在转基因唐鱼中遗传和表达的稳定性分析%Inheritance and expression stability of red fluorescent protein gene in transgenic (Tanichthys albonubes)

    Institute of Scientific and Technical Information of China (English)

    姜鹏; 白俊杰; 简清

    2012-01-01

    The stability of the red fluorescent protein (RFP) transgenes is important for application of the transgenic Tanichthys albonubes line.This paper investigated comparatively the inheritance and expression of RFP gene in different transgenic T. albonubes generations. Under the observation of fluorescence microscope, RFP gene was expressed in almost all body tissues, and both F6 and F10 generations had a similar expression pattern. Results of self-cross and test-cross breeding experiments of transgenic F6 and F10 individuals indicated that segregation of RFP gene still followed Mendelian single-gene inheritance, and their transgenic progenies had a uniform phenotype. The regions including myosin light polypeptide 2 promoter, RFP gene coding sequence and transgene flanking junctions were amplified by PCR from genomic DNA of F2, F6 and F10 generations. Sequence alignment analysis showed that these important regions were well preserved in transgenic fish, and any kind of alteration at nucleotide level was not found. These results suggested that, RFP gene in transgenic T. albonubes maintained stable inheritance and expression through successive generations.%为了评估转红色荧光蛋白(red fluorescent protein,RFP)基因唐鱼新品系的稳定性,研究了RFP基因在不同世代转基因唐鱼中的遗传和表达情况.荧光显微镜下观察显示,RFP基因在F6和F10代所检测的组织器官中均有表达,并且两个世代间相同组织部位的表达水平相似.F6和F10代个体分别配对繁殖实验表明,RFP基因在转基因唐鱼后代中的遗传仍然符合孟德尔分离规律,而且培育出的转基因个体表型特征无显著差异.利用PCR技术在F2、F6和F10代转基因唐鱼基因组中扩增外源性肌球蛋白轻链2启动子、RFP基因编码区和整合位点上下游侧翼区域(片段总长度为4 883 bp),测序结果显示,3个世代间的外源基因序列完全相同,没有发生碱基缺失或突变等现象.研究表明,

  11. Toward an ideal animal model to trace donor cell fates after stem cell therapy: production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene.

    Science.gov (United States)

    Hsiao, F S H; Lian, W S; Lin, S P; Lin, C J; Lin, Y S; Cheng, E C H; Liu, C W; Cheng, C C; Cheng, P H; Ding, S T; Lee, K H; Kuo, T F; Cheng, C F; Cheng, W T K; Wu, S C

    2011-11-01

    The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

  12. Fluorescent Screening of Transgenic Arabidopsis Seeds without Germination1

    Science.gov (United States)

    Wei, Shu; Bravdo, Ben-Ami; Shoseyov, Oded

    2004-01-01

    In this paper, we describe a reliable method for the screening and selection of Arabidopsis transgenic seeds within minutes without germination. Expression of the Aspergillus niger β-glucosidase gene BGL1 in the plant's endoplasmic reticulum was used as a visual marker, together with 4-methylumbelliferyl-β-d-glucopyranoside (MUGluc) as a substrate. Subsequent to incubation in a solution of MUGluc at room temperature for 2 to 15 min, transgenic seeds expressing BGL1 demonstrated a distinct fluorescent signal under UV light. Optimal screening conditions at room temperature were achieved between 75 and 450 μm MUGluc, at a pH of 2.5 to 5.0 and 2 to 5 min of incubation. No significant loss of viability was detected in transgenic seeds that were redried and stored for 45 d after incubation in MUGluc solution for 2 to 150 min. Transgenic plants expressing BGL1 displayed normal phenotypes relative to the wild type. Selection frequency was 3.1% ± 0.34% for the fluorescence selection method, while kanamycin resistant selection resulted in only 0.56% ± 0.13% using the same seed batch. This novel selection method is nondestructive, practical, and efficient, and eliminates the use of antibiotic genes. In addition, the procedure shortens the selection time from weeks to minutes. PMID:15208418

  13. Transgenic mouse model expressing green fluorescent protein for lung cancer%绿色荧光蛋白转基因小鼠肺癌模型的建立

    Institute of Scientific and Technical Information of China (English)

    佘佐亚; 罗殿中; 邝晓聪; 孙忠亮

    2012-01-01

    Objective To establish the lung cancer model of green fluorescent protein (GFP) transgenic BALB/c mouse and observe the expression of green fluorescent protein in the lung tissue after successfully inducing cancer. Methods 4—6 weeks old male and female GFP transgenic mice(n=60) were randomly divided into an experimental group (n=50) and a control group(re=10). The mice were treated with diethylnitrosamine through gavage, 30 mg/kg body weight in distilled water, twice a week for eight consecutive weeks and then the treatment was stopped, the mice were fed for another 12 weeks. The lung tissue was removed for frozen section to observe the GFP expression by fluorescence microscope, stained with hematoxylin and eosin for histopathological examinaion and immunohistochemical analysis of CK7 and CK14 at the 15th week and the 22rd week rspectively. In addition, the tissue fractions were transplanted into the subcutaneous of the nude mice for preparing the tumor-bearing mice. The biofluorescent signals and survival of xenograft were detected in sequential two weeks using in vivo fluorescent imaging system. Results The lung cancer model of fluorescent BALB/c mouse was successfully established by 0.3% DENA gavage. The inducing rate of tumor was 65% at the 22rd week after treatment with diethylnitrosamine. Tumors manifested positive expression of CK14 and negative expression of CK7, none of which combined with extrapulmonary metastasis. Frozen sections and subcutaneous tumors strongly expressed green fluorescent protein. Conclusion Long-term and low-dose diethylnitrosamine exposure can induce BABL/c mouse lung cancer. The cells of lung cancer can present stable high-level GFP expression in vivo.%目的 建立绿色荧光蛋白(green fluorescent protein,GFP)转基因小鼠肺癌模型,观察诱癌后肺组织细胞GFP的表达情况.方法 将60只4~6周龄SPF级BALB/c绿色荧光蛋白转基因小鼠随机分为实验(30 mg/kg二甲基亚硝胺)组(50

  14. Fluorescent transgenic zebrafish as a biosensor for growth-related effects of methyl parathion.

    Science.gov (United States)

    Almeida, Daniela Volcan; Vaz, Bernardo; Azevedo Figueiredo, Márcio; Junior, Antonio Sergio Varela; Marins, Luis Fernando

    2014-07-01

    Transgenic fish models are potential alternative subjects in toxicological studies, since they can provide in vivo information on the deleterious effects of different substances. Here, we used a transgenic zebrafish (Danio rerio) lineage, which expresses a destabilized fluorescent protein (DsRED) driven by the myosin light chain promoter (Mylz2), in order to propose a new research tool for environmental biomonitoring. For validating the MYO-RED lineage, we exposed fish to the organophosphorated pesticide methyl parathion (MP). The effect of MP on fish growth was assessed by evaluating weight, length, condition factor and muscle fiber diameter. All factors suffered reduction at both tested concentrations (0.13μM and 13μM of MP). Similarly, fluorescence intensity decreased in a concentration-dependent manner, suggesting muscle protein catabolism. However, DsRED gene expression lowered only at the higher MP concentration. Results indicate that the MYO-RED transgenic zebrafish is an interesting model for detecting the growth-related effects of pollutants. Destabilized proteins such as reporter genes are apparently sensitive biomarkers, since effects were observed even at the lower, environmentally acceptable concentration. Therefore, this transgenic fish is a promising candidate model for sensitive, fast, and easy environmental monitoring.

  15. Through the Looking Glass: Visualizing Leukemia Growth, Migration, and Engraftment Using Fluorescent Transgenic Zebrafish

    Directory of Open Access Journals (Sweden)

    Finola E. Moore

    2012-01-01

    Full Text Available Zebrafish have emerged as a powerful model of development and cancer. Human, mouse, and zebrafish malignancies exhibit striking histopathologic and molecular similarities, underscoring the remarkable conservation of genetic pathways required to induce cancer. Zebrafish are uniquely suited for large-scale studies in which hundreds of animals can be used to investigate cancer processes. Moreover, zebrafish are small in size, optically clear during development, and amenable to genetic manipulation. Facile transgenic approaches and new technologies in gene inactivation have provided much needed genomic resources to interrogate the function of specific oncogenic and tumor suppressor pathways in cancer. This manuscript focuses on the unique attribute of labeling leukemia cells with fluorescent proteins and directly visualizing cancer processes in vivo including tumor growth, dissemination, and intravasation into the vasculature. We will also discuss the use of fluorescent transgenic approaches and cell transplantation to assess leukemia-propagating cell frequency and response to chemotherapy.

  16. FLUORESCENT TRANSGENIC FISH IN PERU: BIOSAFETY AND RISK ANALYSIS PENDING

    Directory of Open Access Journals (Sweden)

    Scotto, Carlos

    2010-07-01

    Full Text Available Transgenesis involves processes of molecular genetic manipulation of DNAwhich seeks to "introduce genes" of interest from one organism into the genetic material of another to obtain goods or services. The resulting organism is called a Genetically Modified Organism or GMO. It shows the first case of transgenic fluorescent fish as a real example of GMOs existing in Peru. Reproduction and hybridization in confined environments, provide new approaches to biosecurity decision-makers about this new technological contribution to the task of Peru.

  17. 非离子态氨对转红色荧光蛋白基因唐鱼的毒性研究%Effects of un-ionized ammonia on toxicity in red fluorescent protein transgenic Tanichthys albonubes

    Institute of Scientific and Technical Information of China (English)

    吴晗; 白俊杰; 姜鹏

    2013-01-01

    水体中氨氮是影响鱼类生存的重要环境胁迫因子.利用静水法测定水体中非离子态氨对转红色荧光蛋白基因唐鱼(Tanichthys albonubes)和野生唐鱼的急性毒性和慢性效应.急性毒性实验结果显示,转基因唐鱼的非离子态氨24、48、72、96 h半数致死浓度(LC50) (2.43、2.12、1.82、1.72 mg·L-1)分别比野生唐鱼相应时间的半数致死浓度(LC50) (2.40、2.10、1.76、1.67 mg·L-1)略高,但无显著性差异;慢性毒性实验结果显示,在不同浓度非离子态氨(0.02、0.18、0.35、0.53、0.70 mg·L-1)胁迫下,转基因唐鱼和野生唐鱼的死亡率无显著差异.研究结果可为评价转红色荧光蛋白基因唐鱼潜在生态风险提供参考依据.%Ammonia is an important environmental stress factor for fish survival. In this paper, the acute and chronic toxicity of un-ionized ammonia to red fluorescent protein transgenic Tanichthys albonubes and wild T. albonubes were studied by a static bioassay. The results showed that: under the acute toxicity test,the 24,48, 72 and 96 h median lethal concentrations (LC50) for un-ionized ammonia were slightly higher in transgenic T. albonubes (2.43,2. 12,1.82,1.72 mg·L-1,respectively) than in wild T.albonubes (2. 40,2. 10,1. 76, 1.67 mg·L-1, respectively), but there showed no significant difference.Moreover, there was also no significant difference in the mortality of transgenic and wild T.albonubes under different un-ionized ammonia concentrations (0.02,0. 18,0. 35,0. 53,0. 70 mg·L-1) in chronic toxicity test The results are useful for evaluating potential environmental risk of red fluorescent protein transgenic T. albonubes.

  18. The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation

    Directory of Open Access Journals (Sweden)

    Rescan Pierre-Yves

    2010-05-01

    Full Text Available Abstract Background Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f promoter, and cultivated genetically modified myogenic cells derived from these fish. Results In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an in vitro myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this in vitro myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms. Conclusions We produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This in vitro myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.

  19. Chronic oestradiol reduces the dendritic spine density of KNDy (kisspeptin/neurokinin B/dynorphin) neurones in the arcuate nucleus of ovariectomised Tac2-enhanced green fluorescent protein transgenic mice.

    Science.gov (United States)

    Cholanian, M; Krajewski-Hall, S J; McMullen, N T; Rance, N E

    2015-04-01

    Neurones in the arcuate nucleus that express neurokinin B (NKB), kisspeptin and dynorphin (KNDy) play an important role in the reproductive axis. Oestradiol modulates the gene expression and somatic size of these neurones, although there is limited information available about whether their dendritic structure, a correlate of cellular plasticity, is altered by oestrogens. In the present study, we investigated the morphology of KNDy neurones by filling fluorescent neurones in the arcuate nucleus of Tac2-enhanced green fluorescent protein (EGFP) transgenic mice with biocytin. Filled neurones from ovariectomised (OVX) or OVX plus 17β-oestradiol (E2)-treated mice were visualised with anti-biotin immunohistochemistry and reconstructed in three dimensions with computer-assisted microscopy. KNDy neurones exhibited two primary dendrites, each with a few branches confined to the arcuate nucleus. Quantitative analysis revealed that E2 treatment of OVX mice decreased the cell size and dendritic spine density of KNDy neurones. The axons of KNDy neurones originated from the cell body or proximal dendrite and gave rise to local branches that appeared to terminate within the arcuate nucleus. Numerous terminal boutons were also visualised within the ependymal layer of the third ventricle adjacent to the arcuate nucleus. Axonal branches also projected to the adjacent median eminence and exited the arcuate nucleus. Confocal microscopy revealed close apposition of EGFP and gonadotrophin-releasing hormone-immunoreactive fibres within the median eminence and confirmed the presence of KNDy axon terminals in the ependymal layer of the third ventricle. The axonal branching pattern of KNDy neurones suggests that a single KNDy neurone could influence multiple arcuate neurones, tanycytes in the wall of the third ventricle, axon terminals in the median eminence and numerous areas outside of the arcuate nucleus. In parallel with its inhibitory effects on electrical excitability, E2 treatment

  20. A double built-in containment strategy for production of recombinant proteins in transgenic rice.

    Science.gov (United States)

    Zhang, Xianwen; Wang, Dongfang; Zhao, Sinan; Shen, Zhicheng

    2014-01-01

    Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly. However, a major concern over the bioreactor transgenic rice is the risk of its unintended spreading into environment and into food or feed supplies. Here we report a mitigating method to prevent unwanted transgenic rice spreading by a double built-in containment strategy, which sets a selectively termination method and a visual tag technology in the T-DNA for transformation. We created transgenic rice with an inserted T-DNA that harbors a human proinsulin gene fused with the far-red fluorescent protein gene mKate_S158A, an RNAi cassette suppressing the expression of the rice bentazon detoxification enzyme CYP81A6, and an EPSPS gene as the selection marker for transformation. Herbicide spray tests indicated that such transgenic rice plants can be killed selectively by a spray of bentazon at regular field application dosage for rice weed control. Moreover, the transgenic rice seeds were bright red in color due to the fused far-red fluorescent protein, and could be easily visualized under daylight by naked eyes. Thus, the transgenic rice plants reported in this study could be selectively killed by a commonly used herbicide during their growth stage, and their seeds may be detected visually during processing and consumption after harvest. This double built-in containment strategy may greatly enhance the confinement of the transgenic rice.

  1. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  2. Transgenic rats with green, red, and blue fluorescence: powerful tools for bioimaging, cell trafficking, and differentiation

    Science.gov (United States)

    Murakami, Takashi; Kobayashi, Eiji

    2005-04-01

    The rat represents a perfect animal for broadening medical experiments, because its physiology has been well understood in the history of experimental animals. In addition, its larger body size takes enough advantage for surgical manipulation, compared to the mouse. Many rat models mimicking human diseases, therefore, have been used in a variety of biomedical studies including physiology, pharmacology, transplantation, and immunology. In an effort to create the specifically designed rats for biomedical research and regenerative medicine, we have developed the engineered rat system on the basis of transgenic technology and succeeded in establishing various transgenic rat strains. The transgenic rats with green fluorescent protein (GFP) were generated in the two different strains (Wistar and Lewis), in which GFP is driven under the chicken beta-actin promoter and cytomegalovirus enhancer (CAG promoter). Their GFP expression levels were different in each organ, but the Lewis line expressed GFP strongly and ubiquitously in most of the organs compared with that of Wistar. For red fluorescence, DsRed2 was transduced to the Wistar rats: one line specifically expresses DsRed2 in the liver under the mouse albumin promoter, another is designed for the Cre/LoxP system as the double reporter rat (the initial DsRed2 expression turns on GFP in the presence of Cre recombinase). LacZ-transgenic rats represent blue color, and LacZ is driven the CAG (DA) or ROSA26 promoter (Lewis). Our unique transgenic rats" system highlights the powerful performance for the elucidation of many cellular processes in regenerative medicine, leading to innovative medical treatments.

  3. Chromosomal Location and Expression of Green Fluorescent Protein (gfp) Gene in Microspore Derived Transgenic Barley (Hordeum vulgare L.)%转基因大麦中gfp基因的染色体位置及其表达

    Institute of Scientific and Technical Information of China (English)

    陈建民; Carlson A R; 万建民; Kasha K J

    2003-01-01

    通过对大麦小孢子进行基因枪轰击获得4株转绿色荧光蛋白基因(gfp)的植株(A、C、D、E),以gfp基因为探针进行荧光原位杂交(FISH)研究转化植株中转基因插入位置和基因表达.4个株系在染色体7L(5HL)的不同位置都有一个插入点,而E株系在染色体5S(7HS)还有第2个插入点.所有的转基因T0代植株都是半合子并在T1、T2代发生分离.D株系GFP未表达,但FISH和PCR分析表明gfp基因已成功插入其染色体.各株系在根尖和花粉中的GFP表达水平不同:C株系在花粉表达强而在根尖表达中等;A株系在花粉中等表达而在根尖表达较淡;E株系则在根尖高表达,花粉中等表达.A和C株系在根尖和花粉的GFP分离都表现单位点特性,而E株系的根尖分离表现重叠作用(15∶1)特征,但在花粉中表达GFP的频率低.PCR结果和3个分离株系的根尖表达结果一致.D和E株系的GFP表达不正常可能和gfp基因插入位置或基因的结构有关.%Four doubled haploid barley lines (A,C,D,E) derived from gfp (green fluorescent protein) transformation and selection following particle bombardment of microspores were studied for gene expression pattern and the location of genome inserts.The integration sites were detected by fluorescence in situ hybridization (FISH) using the gfp plasmid DNA as a probe.Plants from events A,C,D and E all have a single insert site on chromosome 7L(5HL) at different locations while line E has a second insert site on chromosome 5S(7HS).All original transgenic plants were hemizygous for the transgenes and segregated in the T1 and T2 generations.Although line D had no GFP expression,FISH and PCR could detect gfp gene on its chromosome in transformed plants.Expression levels of GFP varied with lines and tissues examined.Plants from line C showed good expression in pollen and an intermediate level in root tips.Plants from A have intermediate expression of GFP in the pollen and light expression in the

  4. A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay.

    Science.gov (United States)

    Bou, Gerelchimeg; Sun, Mingju; Lv, Ming; Zhu, Jiang; Li, Hui; Wang, Juan; Li, Lu; Liu, Zhongfeng; Zheng, Zhong; He, Wenteng; Kong, Qingran; Liu, Zhonghua

    2014-08-01

    For efficient transgenic herd expansion, only the transgenic animals that possess the ability to transmit transgene into next generation are considered for breeding. However, for transgenic pig, practically lacking a pre-breeding screening program, time, labor and money is always wasted to maintain non-transgenic pigs, low or null transgenic transmission pigs and the related fruitless gestations. Developing a pre-breeding screening program would make the transgenic herd expansion more economical and efficient. In this technical report, we proposed a three-step pre-breeding screening program for transgenic boars simply through combining the fluorescence in situ hybridization (FISH) assay with the common pre-breeding screening workflow. In the first step of screening, combined with general transgenic phenotype analysis, FISH is used to identify transgenic boars. In the second step of screening, combined with conventional semen test, FISH is used to detect transgenic sperm, thus to identify the individuals producing high quality semen and transgenic sperm. In the third step of screening, FISH is used to assess the in vitro fertilization embryos, thus finally to identify the individuals with the ability to produce transgenic embryos. By this three-step screening, the non-transgenic boars and boars with no ability to produce transgenic sperm or transgenic embryos would be eliminated; therefore only those boars could produce transgenic offspring are maintained and used for breeding and herd expansion. It is the first time a systematic pre-breeding screening program is proposed for transgenic pigs. This program might also be applied in other transgenic large animals, and provide an economical and efficient strategy for herd expansion.

  5. Anatomy of the pectoral and forelimb muscles of wildtype and green fluorescent protein-transgenic axolotls and comparison with other tetrapods including humans: a basis for regenerative, evolutionary and developmental studies

    Science.gov (United States)

    Diogo, R; Tanaka, E M

    2012-01-01

    The axolotl Ambystoma mexicanum is one of the most used model organisms in evolutionary, developmental and regenerative studies, particularly because it can reconstitute a fully functional and complete forelimb/hindlimb. Surprisingly, there is no publication that describes all the pectoral and forelimb muscles of this species or provides a comparative framework between these muscles and those of other model organisms and of modern humans. In the present paper we describe and illustrate all these muscles in A. mexicanum and provide the first report about the myology of adults of a model organism that is based on analyses and dissections of both wildtype animals and transgenic animals that express green fluorescent protein (GFP) in muscle fibers. On the one hand, the inclusion of GFP-transgenic animals allows us to show the muscles as more commonly seen, and thus easier to understand, by current developmental and regenerative biologists. On the other hand, by including wildtype and GFP-transgenic animals and by visualizing these latter animals with and without a simultaneous transmission laser light, we were able to obtain a more complete and clearer understanding of the exact limit of the fleshy and tendinous parts of the muscles and their specific connections with the skeletal elements. This in turn allowed us to settle some controversies in previous anatomical and comparative studies. As most developmental, regenerative and evolutionary biologists are interested in comparing their observations of A. mexicanum with observations in other model organisms, and ultimately in using this information to increase the understanding of human evolution and medicine, we also provide tables showing the homologies between the pectoral and forelimb muscles of axolotls, of model organisms such as mice, frogs and chicken, and of Homo sapiens. An example illustrating the outcomes of using our methodology and of our observations is that they revealed that, contrary to what is often

  6. Anatomy of the pectoral and forelimb muscles of wildtype and green fluorescent protein-transgenic axolotls and comparison with other tetrapods including humans: a basis for regenerative, evolutionary and developmental studies.

    Science.gov (United States)

    Diogo, R; Tanaka, E M

    2012-12-01

    The axolotl Ambystoma mexicanum is one of the most used model organisms in evolutionary, developmental and regenerative studies, particularly because it can reconstitute a fully functional and complete forelimb/hindlimb. Surprisingly, there is no publication that describes all the pectoral and forelimb muscles of this species or provides a comparative framework between these muscles and those of other model organisms and of modern humans. In the present paper we describe and illustrate all these muscles in A. mexicanum and provide the first report about the myology of adults of a model organism that is based on analyses and dissections of both wildtype animals and transgenic animals that express green fluorescent protein (GFP) in muscle fibers. On the one hand, the inclusion of GFP-transgenic animals allows us to show the muscles as more commonly seen, and thus easier to understand, by current developmental and regenerative biologists. On the other hand, by including wildtype and GFP-transgenic animals and by visualizing these latter animals with and without a simultaneous transmission laser light, we were able to obtain a more complete and clearer understanding of the exact limit of the fleshy and tendinous parts of the muscles and their specific connections with the skeletal elements. This in turn allowed us to settle some controversies in previous anatomical and comparative studies. As most developmental, regenerative and evolutionary biologists are interested in comparing their observations of A. mexicanum with observations in other model organisms, and ultimately in using this information to increase the understanding of human evolution and medicine, we also provide tables showing the homologies between the pectoral and forelimb muscles of axolotls, of model organisms such as mice, frogs and chicken, and of Homo sapiens. An example illustrating the outcomes of using our methodology and of our observations is that they revealed that, contrary to what is often

  7. Transgenic chickens as bioreactors for protein-based drugs.

    Science.gov (United States)

    Lillico, Simon G; McGrew, Michael J; Sherman, Adrian; Sang, Helen M

    2005-02-01

    The potential of using transgenic animals for the synthesis of therapeutic proteins was suggested over twenty years ago. Considerable progress has been made in developing methods for the production of transgenic animals and specifically in the expression of therapeutic proteins in the mammary glands of cows, sheep and goats. Development of transgenic hens for protein production in eggs has lagged behind these systems. The positive features associated with the use of the chicken in terms of cost, speed of development of a production flock and potentially appropriate glycosylation of target proteins have led to significant advances in transgenic chicken models in the past few years.

  8. Simultaneous tracking of movement and gene expression in multiple Drosophila melanogaster flies using GFP and DsRED fluorescent reporter transgenes

    Directory of Open Access Journals (Sweden)

    Tavaré Simon

    2009-04-01

    Full Text Available Abstract Background Fluorescent proteins such as GFP (Green Fluorescent Protein and DsRED (Discosoma sp.Red Fluorescent Protein are often used as reporter molecules for transgene expression in Drosophila and other species. We have recently reported methods that allow simultaneous tracking of animal movement and GFP expression in real time, however the assay was limited to single animals and a single transgene. Numerous studies would be facilitated by methods that allow for assay of multiple animals and multiple transgenes. Findings Here we report an improved fly video tracking system that allows multiple transgenic flies to be tracked simultaneously using visible light, GFP fluorescence and DsRED fluorescence. The movement of multiple flies could be accurately tracked at real time rates, while simultaneously assaying the expression level of two different transgenes marked with GFP and DsRED. The individual flies could be accurately tracked and distinguished even during periods when transgene fluorescence was undetected. For example, characteristic patterns of hsp70 and hsp22 transgene induction could be simultaneously quantified and correlated with animal movement in aging flies, and as groups of flies died due to dessication/starvation. Conclusion The improved methods allow for more efficient assay of the correlation between gene expression, behavior, aging and mortality: multiple animals can be assayed with simultaneous quantification of multiple transgenes using GFP and DsRED fluorescence. These methods should allow for increased flexibility in experimental designs. For example, in the future it should be possible to use gene expression levels to predict remaining life span more accurately, and to quantify gene expression changes caused by interactions between animals in real time.

  9. POLA EKSPRESI GEN ENHANCED GREEN FLUORESCENT PROTEIN PADA EMBRIO DAN LARVA IKAN PATIN SIAM (Pangasianodon hypophthalmus)

    OpenAIRE

    Raden Roro Sri Pudji Sinarni Dewi; Alimuddin Alimuddin; Agus Oman Sudrajat; Komar Sumantadinata; Erma Primanita Hayuningtyas

    2016-01-01

    Penelitian ekspresi sementara (transient expression) dari transgen secara in vivo menggunakan gen reporter berguna untuk mendesain konstruksi gen yang akan digunakan pada penelitian transgenesis. Gen reporter yang umum digunakan dalam penelitian ekspresi sementara transgen adalah gen GFP (green fluorescent protein). Pengamatan gen EGFP (enhanced green fluorescent protein) pada embrio dan larva ikan patin siam (Pangasianodon hypophthalmus) ditujukan untuk mendapatkan informasi mengenai kema...

  10. FLUORESCENCE LIFETIME DISTRIBUTIONS IN PROTEINS

    OpenAIRE

    ALCALA, JR; Gratton, E; PRENDERGAST, FG

    1987-01-01

    The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most ...

  11. Lasing from fluorescent protein crystals.

    Science.gov (United States)

    Oh, Heon Jeong; Gather, Malte C; Song, Ji-Joon; Yun, Seok Hyun

    2014-12-15

    We investigated fluorescent protein crystals for potential photonic applications, for the first time to our knowledge. Rod-shaped crystals of enhanced green fluorescent protein (EGFP) were synthesized, with diameters of 0.5-2 μm and lengths of 100-200 μm. The crystals exhibit minimal light scattering due to their ordered structure and generate substantially higher fluorescence intensity than EGFP or dye molecules in solutions. The magnitude of concentration quenching in EGFP crystals was measured to be about 7-10 dB. Upon optical pumping at 485 nm, individual EGFP crystals located between dichroic mirrors generated laser emission with a single-mode spectral line at 513 nm. Our results demonstrate the potential of protein crystals as novel optical elements for self-assembled, micro- or nano-lasers and amplifiers in aqueous environment.

  12. Going Viral with Fluorescent Proteins.

    Science.gov (United States)

    Costantini, Lindsey M; Snapp, Erik L

    2015-10-01

    Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

  13. Production of recombinant proteins in milk of transgenic and non-transgenic goats

    Directory of Open Access Journals (Sweden)

    Raylene Ramos Moura

    2011-10-01

    Full Text Available Among all the transgenic mammalians produced so far, goats have represented an excellent model of transgenesis when considering the factors such as the market demand for protein, volume of milk produced per lactation and reproductive rate. Various recombinant proteins have been obtained from the transgenic and non-transgenic goats, and among these, human antithrombin, produced by the transgenic goats, was the first recombinant protein of animal origin to be released as a drug for the clinical use in humans. This review reports the aspects inherent to the production of recombinant proteins in the goats, from the production of the animal bioreactors up to the expression of these proteins in their milk.

  14. Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

    Science.gov (United States)

    Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

    2015-01-01

    The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in

  15. Transgenic animal bioreactors in biotechnology and production of blood proteins.

    Science.gov (United States)

    Lubon, H

    1998-01-01

    The regulatory elements of genes used to target the tissue-specific expression of heterologous human proteins have been studied in vitro and in transgenic mice. Hybrid genes exhibiting the desired performance have been introduced into large animals. Complex proteins like protein C, factor IX, factor VIII, fibrinogen and hemoglobin, in addition to simpler proteins like alpha 1-antitrypsin, antithrombin III, albumin and tissue plasminogen activator have been produced in transgenic livestock. The amount of functional protein secreted when the transgene is expressed at high levels may be limited by the required posttranslational modifications in host tissues. This can be overcome by engineering the transgenic bioreactor to express the appropriate modifying enzymes. Genetically engineered livestock are thus rapidly becoming a choice for the production of recombinant human blood proteins.

  16. Effects of Food Supply Level on Survival and Growth of Transgenic Tanichthys albonubes Expressing the Red Fluorescent Protein%食物供应量对转红色荧光蛋白基因唐鱼生存和生长的影响

    Institute of Scientific and Technical Information of China (English)

    樊佳佳; 白俊杰; 简清; 叶星

    2011-01-01

    We assessed fitness of transgenic Tanichthys albonubes to food supply level using 7-days healthy larvge from crossing heterozygous transgenic T. albonubes expressing the red fluorescent protein to non-transgenic T. albonubes. The larvae were randomly divided into three groups: satiation group ( high food supply level), semi-starvation group (medium food supply level) and starvation group (low food supply level) under (25. 0 ± 2. O) ℃. The results showed that: ( 1 ) the mortality rate of 72-days of larvae in satiation group was not significantly different from those in semi-starvation group ( P > O. 05 ), but larvae in starvation group was significant difference from both satiation group and semi-starvation group ( P < 0.01 ). It indicates that food supply level affect the mortality rate of T. albonubes. (2) The red fluorescent protein was initially expression at 30-days larvae with visual inspection. The PCR method was used to make sure that there was no recessive expression in the three treatments at 72-days larval. At this point the number of transgenic and non-transgenic T. albonubes was close to the 1 ∶1 ratio, and consistent with classical Mendelian inheritance model, which indicates that transgenic and non-transgenic T. albonubes are equally effected by food supply level. (3) The body length at 28-, 42-, 57- and 72-days larvae was satiation group > semi-starvation group > starvation group at significant or very significant level (P < O. 05 or P < 0.01 ). The larvae body lenth of 72-days larvae has no significantly between transgenic and non-transgenic ones within the same food supply level group. In summary, the food supply level had similar influence on survival and growth of T. albonubes for both transgenic and nontransgenic. We speculate that transgenic T. albonubes have no competition advantage in the low food availability.%选取雄性杂合子转红色荧光蛋白基因唐鱼(Tanichthys albonubes)(简称:转基因唐鱼)与雌性非转

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

    Science.gov (United States)

    Monteiro, Antónia

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mainak Das Gupta

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Mushroom body miscellanea: transgenic Drosophila strains expressing anatomical and physiological sensor proteins in Kenyon cells.

    Science.gov (United States)

    Pech, Ulrike; Dipt, Shubham; Barth, Jonas; Singh, Priyanka; Jauch, Mandy; Thum, Andreas S; Fiala, André; Riemensperger, Thomas

    2013-01-01

    The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function.

  1. Fluorescent Protein Approaches in Alpha Herpesvirus Research

    Directory of Open Access Journals (Sweden)

    Ian B. Hogue

    2015-11-01

    Full Text Available In the nearly two decades since the popularization of green fluorescent protein (GFP, fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1 and pseudorabies virus (PRV structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

  2. A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants.

    Science.gov (United States)

    Ochiai-Fukuda, Tetsuko; Takahashi-Ando, Naoko; Ohsato, Shuichi; Igawa, Tomoko; Kadokura, Kaori; Hamamoto, Hiroshi; Nakasako, Masayoshi; Kudo, Toshiaki; Shibata, Takehiko; Yamaguchi, Isamu; Kimura, Makoto

    2006-04-20

    Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.

  3. Cell tracking using a photoconvertible fluorescent protein.

    Science.gov (United States)

    Hatta, Kohei; Tsujii, Hitomi; Omura, Tomomi

    2006-01-01

    The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2-8 d.

  4. Transgenic rabbits as therapeutic protein bioreactors and human disease models.

    Science.gov (United States)

    Fan, Jianglin; Watanabe, Teruo

    2003-09-01

    Genetically modified laboratory animals provide a powerful approach for studying gene expression and regulation and allow one to directly examine structure-function and cause-and-effect relationships in pathophysiological processes. Today, transgenic mice are available as a research tool in almost every research institution. On the other hand, the development of a relatively large mammalian transgenic model, transgenic rabbits, has provided unprecedented opportunities for investigators to study the mechanisms of human diseases and has also provided an alternative way to produce therapeutic proteins to treat human diseases. Transgenic rabbits expressing human genes have been used as a model for cardiovascular disease, AIDS, and cancer research. The recombinant proteins can be produced from the milk of transgenic rabbits not only at lower cost but also on a relatively large scale. One of the most promising and attractive recombinant proteins derived from transgenic rabbit milk, human alpha-glucosidase, has been successfully used to treat the patients who are genetically deficient in this enzyme. Although the pronuclear microinjection is still the major and most popular method for the creation of transgenic rabbits, recent progress in gene targeting and animal cloning has opened new avenues that should make it possible to produce transgenic rabbits by somatic cell nuclear transfer in the future. Based on a computer-assisted search of the studies of transgenic rabbits published in the English literature here, we introduce to the reader the achievements made thus far with transgenic rabbits, with emphasis on the application of these rabbits as human disease models and live bioreactors for producing human therapeutic proteins and on the recent progress in cloned rabbits.

  5. Fluorescent sensors based on bacterial fusion proteins

    Science.gov (United States)

    Prats Mateu, Batirtze; Kainz, Birgit; Pum, Dietmar; Sleytr, Uwe B.; Toca-Herrera, José L.

    2014-06-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

  6. "Color Timer" mice: visualization of neuronal differentiation with fluorescent proteins.

    Science.gov (United States)

    Kanki, Hiroaki; Shimabukuro, Marilia Kimie; Miyawaki, Atsushi; Okano, Hideyuki

    2010-02-02

    The molecular mechanisms governing the differentiation of neural stem cells (NSCs) into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg) mice in which production of the fluorescent protein Kusabira-Orange (KOr) is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to green. This two-color initial

  7. Highlights of the optical highlighter fluorescent proteins.

    Science.gov (United States)

    Patterson, G H

    2011-07-01

    The development of super-resolution microscopy techniques using molecular localization, such as photoactivated localization microscopy, fluorescence photoactivated localization microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy with independent running acquisition and many others, has heightened interest in molecules that will be grouped here into a category referred to as 'optical highlighter' fluorescent proteins. This review will survey many of the advances in development of fluorescent proteins for optically highlighting sub-populations of fluorescently labelled molecules.

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

    OpenAIRE

    Mainak Das Gupta; Sam Kok Sim Chan; Antónia Monteiro

    2015-01-01

    Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescen...

  9. Quantitation of Brown Adipose Tissue Perfusion in Transgenic Mice Using Near-Infrared Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Akira Nakayama

    2003-01-01

    Full Text Available Brown adipose tissue (BAT; brown fat is the principal site of adaptive thermogenesis in the human newborn and other small mammals. Of paramount importance for thermogenesis is vascular perfusion, which controls the flow of cool blood in, and warmed blood out, of BAT. We have developed an optical method for the quantitative imaging of BAT perfusion in the living, intact animal using the heptamethine indocyanine IR-786 and near-infrared (NIR fluorescent light. We present a detailed analysis of the physical, chemical, and cellular properties of IR-786, its biodistribution and pharmacokinetics, and its uptake into BAT. Using transgenic animals with homozygous deletion of Type II iodothyronine deiodinase, or homozygous deletion of uncoupling proteins (UCPs 1 and 2, we demonstrate that BAT perfusion can be measured noninvasively, accurately, and reproducibly. Using these techniques, we show that UCP 1/2 knockout animals, when compared to wild-type animals, have a higher baseline perfusion of BAT but a similar maximal response to β3-receptor agonist. These results suggest that compensation for UCP deletion is mediated, in part, by the control of BAT perfusion. Taken together, BAT perfusion can now be measured noninvasively using NIR fluorescent light, and pharmacological modulators of thermogenesis can be screened at relatively high throughput in living animals.

  10. Fluorescence dynamics of green fluorescent protein in AOT reversed micelles

    NARCIS (Netherlands)

    Uskova, M.A.; Borst, J.W.; Hink, M.A.; Hoek, van A.; Schots, A.; Klyachko, N.L.; Visser, A.J.W.G.

    2000-01-01

    We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the

  11. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    NARCIS (Netherlands)

    Shaner, Nathan C; Campbell, Robert E; Steinbach, Paul A; Giepmans, Ben N G; Palmer, Amy E; Tsien, Roger Y

    2004-01-01

    Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red

  12. An improved BAC transgenic fluorescent reporter line for sensitive and specific identification of striatonigral medium spiny neurons

    Directory of Open Access Journals (Sweden)

    Kristen eAde

    2011-06-01

    Full Text Available The development of BAC transgenic mice expressing promoter-specific fluorescent reporter proteins has been a great asset for neuroscience by enabling detection of neuronal subsets in live tissue. For the study of basal ganglia physiology, reporters driven by type 1 and 2 dopamine receptors have been particularly useful for distinguishing the two classes of striatal projection neurons - striatonigral and striatopallidal. However, emerging evidence suggests that some of the transgenic reporter lines may have suboptimal features. The ideal transgenic reporter line should (1 express a reporter with high sensitivity and specificity for detecting the cellular subset of interest and that does not otherwise alter the biology of the cells in which it is expressed, and (2 involve a genetic manipulation that does not cause any additional genetic effects other than expression of the reporter. Here we introduce a new BAC transgenic reporter line, Drd1a-tdTomato line 6, with features that approximate these ideals, offering substantial benefits over existing lines. In this study, we investigate the integrity of dopamine-sensitive behaviors and test the sensitivity and specificity of tdTomato fluorescence for identifying striatonigral projection neurons in mice. Behaviorally, hemizygous Drd1a-tdTomato line 6 mice are similar to littermate controls; while hemizygous Drd2-EGFP mice are not. In characterizing the sensitivity and specificity of line 6 mice, we find that both are high. The results of this characterization indicate that line 6 Drd1a-tdTomato+/- mice offer a useful alternative approach to identify both striatonigral and striatopallidal neurons in a single transgenic line with a high degree of accuracy.

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

    Institute of Scientific and Technical Information of China (English)

    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

    2012-01-01

    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

  14. Fluorescence-tagged transgenic lines reveal genetic defects in pollen growth--application to the eIF3 complex.

    Directory of Open Access Journals (Sweden)

    Bijoyita Roy

    Full Text Available BACKGROUND: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3 cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis. METHODOLOGY/PRINCIPAL FINDINGS: Fluorescence tagged lines (FTLs harbor a transgenic fluorescent protein gene (XFP expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. CONCLUSION/SIGNIFICANCE: eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.

  15. Producing proteins in transgenic plants and animals.

    Science.gov (United States)

    Larrick, J W; Thomas, D W

    2001-08-01

    The requirement for large quantities of therapeutic proteins has fueled interest in the production of recombinant proteins in plants and animals. The first commercial products to be made in this way have experienced much success, and it is predicted that in the future a plethora of protein products will be made using these 'natural' bioreactors.

  16. Generation of a fluorescent transgenic zebrafish for detection of environmental estrogens

    Energy Technology Data Exchange (ETDEWEB)

    Chen Hao; Hu Jingying; Yang Jian; Wang Yuexiang; Xu Hui; Jiang Qiu; Gong Yuebo; Gu Yinliang [Department of Molecular Genetics, Shanghai Medical School and Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, 200032 (China); Song Houyan, E-mail: hysong@shmu.edu.cn [Department of Molecular Genetics, Shanghai Medical School and Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, 200032 (China)

    2010-01-21

    To establish a novel in vivo test system for rapid detection of environmental estrogens, an ere-zvtg1: gfp transgenic zebrafish line has been generated. In this transgenic line, under control conditions, GFP was exclusively expressed in the liver of mature adult female fish. Male and larval transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17-{alpha}-ethynylestradiol (EE{sub 2}). Concurrent accumulation of zvtg1 and gfp mRNAs in embryos and larvae after EE{sub 2} exposure was observed, which indicated that the expression of gfp transgene was driven by the zvtg1 promoter. Green fluorescence was first observed in the liver at 53, 74, 100 or 131 h post-fertilization (hpf) after exposure to 100, 10, 1 or 0.1 ng/L EE{sub 2} from 1 to 2 cell stage, respectively. As for mature male transgenic zebrafish, green fluorescence was observed after exposure to 100, 10, 1 or 0.1 ng/L EE{sub 2} for 2, 3, 4 or 7 days, respectively; as for mature female, fluorescence was increased after exposure to relatively high concentrations of EE{sub 2} (10 and 100 ng/L). Green fluorescence in the liver was increased with prolonging of exposure time and was repeatedly induced after removal and re-addition of EE{sub 2}. We also demonstrated that GFP expression could be induced by other estrogenic compounds, including {beta}-estradiol (E{sub 2}, 0.1 {mu}g/L), cadmium chloride (CdCl{sub 2}, 10 {mu}g/L), zearalenone (50 {mu}g/L), estriol (E{sub 3}, 1 {mu}g/L), diethylstilbestrol (DES, 50 ng/L) bisphenol A (BPA, 1 mg/L) but not by weakly estrogenic compounds such as nonylphenol (NP, up to 10 mg/L), or non-estrogenic steroid hormones such as progesterone (up to 100 mg/L) and 17-hydroxysteroid (up to 50 mg/L). These data suggest the transgenic zebrafish is sensitive and specific for detection of estrogenic compounds. Because the observed-effect concentrations are as low as those of environment and the observed-effect exposure times are very short

  17. "Color Timer" mice: visualization of neuronal differentiation with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Kanki Hiroaki

    2010-02-01

    Full Text Available Abstract The molecular mechanisms governing the differentiation of neural stem cells (NSCs into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg mice in which production of the fluorescent protein Kusabira-Orange (KOr is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to

  18. Fluorescent protein integrated white LEDs for displays

    Science.gov (United States)

    Press, Daniel Aaron; Melikov, Rustamzhon; Conkar, Deniz; Nur Firat-Karalar, Elif; Nizamoglu, Sedat

    2016-11-01

    The usage time of displays (e.g., TVs, mobile phones, etc) is in general shorter than their functional life time, which worsens the electronic waste (e-waste) problem around the world. The integration of biomaterials into electronics can help to reduce the e-waste problem. In this study, we demonstrate fluorescent protein integrated white LEDs to use as a backlight source for liquid crystal (LC) displays for the first time. We express and purify enhanced green fluorescent protein (eGFP) and monomeric Cherry protein (mCherry), and afterward we integrate these proteins as a wavelength-converter on a blue LED chip. The protein-integrated backlight exhibits a high luminous efficacy of 248 lm/Wopt and the area of the gamut covers 80% of the NTSC color gamut. The resultant colors and objects in the image on the display can be well observed and distinguished. Therefore, fluorescent proteins show promise for display applications.

  19. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  20. Genotype-independent transmission of transgenic fluorophore protein by boar spermatozoa.

    Directory of Open Access Journals (Sweden)

    Wiebke Garrels

    Full Text Available Recently, we generated transposon-transgenic boars (Sus scrofa, which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring. The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.

  1. Transgenic soybeans and soybean protein analysis: an overview.

    Science.gov (United States)

    Natarajan, Savithiry; Luthria, Devanand; Bae, Hanhong; Lakshman, Dilip; Mitra, Amitava

    2013-12-04

    To meet the increasing global demand for soybeans for food and feed consumption, new high-yield varieties with improved quality traits are needed. To ensure the safety of the crop, it is important to determine the variation in seed proteins along with unintended changes that may occur in the crop as a result various stress stimuli, breeding, and genetic modification. Understanding the variation of seed proteins in the wild and cultivated soybean cultivars is useful for determining unintended protein expression in new varieties of soybeans. Proteomic technology is useful to analyze protein variation due to various stimuli. This short review discusses transgenic soybeans, different soybean proteins, and the approaches used for protein analysis. The characterization of soybean protein will be useful for researchers, nutrition professionals, and regulatory agencies dealing with soy-derived food products.

  2. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  3. Photochromicity and fluorescence lifetimes of green fluorescent protein

    OpenAIRE

    1999-01-01

    The green fluorescent protein (GFP) of the bioluminescent jellyfish Aequorea and its mutants have gained widespread usage as an indicator of structure and function within cells. Proton transfer has been implicated in the complex photophysics of the wild-type molecule, exhibiting a protonated A species excited at 400 nm, and two deprotonated excited-state species I* and B* with red-shifted excitation similar to 475 nm. Photochromicity between the protonated and deprotonated species has been re...

  4. Subcellular localization of the voltage-gated potassium channels Kv3.1b and Kv3.3 in the cerebellar dentate nucleus of glutamic acid decarboxylase 67-green fluorescent protein transgenic mice.

    Science.gov (United States)

    Alonso-Espinaco, V; Elezgarai, I; Díez-García, J; Puente, N; Knöpfel, T; Grandes, P

    2008-09-09

    Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD- and GAD+ dentate neurons of glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD- and GAD+ dentate neurons. The vast majority of GAD- somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD- axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD- somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD- cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD- and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD- and GAD+ neurons in the dentate nucleus.

  5. Conjugation of fluorescent proteins with DNA oligonucleotides.

    Science.gov (United States)

    Lapiene, Vidmantas; Kukolka, Florian; Kiko, Kathrin; Arndt, Andreas; Niemeyer, Christof M

    2010-05-19

    This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E(2)GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable site-selective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.

  6. Molecular farming of industrial proteins from transgenic maize.

    Science.gov (United States)

    Hood, E E; Kusnadi, A; Nikolov, Z; Howard, J A

    1999-01-01

    Recombinant egg white avidin and bacterial B-glucuronidase (GUS) from transgenic maize have been commercially produced. High levels of expression were obtained in seed by employing the ubiquitin promoter from maize. The recombinant proteins had activities that were indistinguishable from their native counterparts. We have illustrated that down-stream activities in the production of these recombinant proteins, such as stabilizing the germplasm and processing for purification, were accomplished without any major obstacles. Avidin (A8706) and GUS (G2035) are currently marketed by Sigma Chemical Co.

  7. Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.

    Science.gov (United States)

    Hermiston, M L; Latham, C B; Gordon, J I; Roth, K A

    1992-09-01

    To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.

  8. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    Directory of Open Access Journals (Sweden)

    Hojman Pernille

    2009-04-01

    Full Text Available Abstract DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

  9. Production of a transgenic mosquito expressing circumsporozoite protein, a malarial protein, in the salivary gland of Anopheles stephensi (Diptera: Culicidae).

    Science.gov (United States)

    Matsuoka, Hiroyuki; Ikezawa, Tsunetaka; Hirai, Makoto

    2010-08-01

    We are producing a transgenic mosquito, a flying syringe, to deliver a vaccine protein to human beings via the saliva the mosquito deposits in the skin while biting. The mosquito produces a vaccine protein in the salivary gland (SG) and deposits the protein into the host's skin when it takes the host's blood. We chose circumsporozoite protein (CSP), currently the most promising malaria vaccine candidate, to be expressed in the SG of Anopheles stephensi. To transform the mosquitoes, plasmid containing the CSP gene under the promoter of female SG-specific gene, as well as the green fluorescent protein (GFP) gene under the promoter of 3xP3 as a selection marker in the eyes, was injected into more than 400 eggs. As a result, five strains of GFP-expressing mosquitoes were established, and successful CSP expression in the SG was confirmed in one strain. The estimated amount of CSP in the SG of the strain was 40 ng per mosquito. We allowed the CSP-expressing mosquitoes to feed on mice to induce the production of anti-CSP antibody. However, the mice did not develop anti-CSP antibody even after transgenic mosquitoes had bitten them several times. We consider that CSP in the SG was not secreted properly into the saliva. Further techniques and trials are required in order to realize vaccine-delivering mosquitoes.

  10. Versatile protein tagging in cells with split fluorescent protein

    OpenAIRE

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A.; Ishikawa, Hiroaki; Leonetti, Manuel D.; Marshall, Wallace F.; Weissman, Jonathan S.; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respec...

  11. Diversity and evolution of coral fluorescent proteins.

    Directory of Open Access Journals (Sweden)

    Naila O Alieva

    Full Text Available GFP-like fluorescent proteins (FPs are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae and pink chromoprotein from Stylophora pistillata (Pocilloporidae, both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria. The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of

  12. Production and purification of two recombinant proteins from transgenic corn.

    Science.gov (United States)

    Kusnadi, A R; Hood, E E; Witcher, D R; Howard, J A; Nikolov, Z L

    1998-01-01

    This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed. The avidin and gus genes were stably integrated in the genome and expressed over seven generations. The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively. Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter. The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity. The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week. The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels. Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.

  13. Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

    Directory of Open Access Journals (Sweden)

    Chan Anthony WS

    2008-02-01

    Full Text Available Abstract Background Alzheimer's disease (AD is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD. Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP. Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated. Results Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP. Conclusion This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue

  14. Breeding and reproduction of transgenic fish fluorescent in captivity: a prospective scope

    Directory of Open Access Journals (Sweden)

    Carlos Scotto Espinoza

    2012-03-01

    Full Text Available The introduction, breeding and hybridization of fluorescent transgenic fish in Peruvian territory has been going since 2006 as demonstrated in laboratory tests reported in this article. He has spoken to Peru Law No. 29811 establishing the moratorium on the production of income and living modified organisms into the country for a period of 10 years for cultivation or breeding, including water, to be released into the environment. The paper shows the existence of aquatic LMOs that are being propagated commercially by aquarists and / or ornamental fish farmers out of control and could potentially be released into the environment. However, preliminary results on the other side could initiate a first analysis and risk management at the laboratory and / or under controlled conditions with these organisms (zebra fish case that could provide the basis for future decision making relevant to the introduction of GMOs for any scientific purpose, commercial, environmental or other national interest not yet glimpsed.

  15. TRANSGENIC STRATEGY FOR IDENTIFYING SYNAPTIC CONNECTIONS IN MICE BY FLUORESCENCE COMPLEMENTATION (GRASP

    Directory of Open Access Journals (Sweden)

    Masahito eYamagata

    2012-02-01

    Full Text Available In the "GFP reconstitution across synaptic partners" (GRASP method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method.

  16. CO2 Exchange and Chlorophyll Fluorescence of Phosphoenolpyruvate Carboxylase Transgenic Rice Pollen Lines

    Institute of Scientific and Technical Information of China (English)

    Li-Li Ling; Hong-Hui Lin; Ben-Hua Ji; De-Mao Jiao

    2006-01-01

    To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice(Oryza sativa L.) hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase (PEPC) transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and transferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield (AQY), photochemical efficiency of photosystem Ⅱ and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or maiate, or phosphoenolpyruvate.The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.

  17. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  18. Real-time immuno-PCR: an approach for detection of trace amounts of transgenic proteins.

    Science.gov (United States)

    Kumar, Rajesh; Sinha, Rajeshwar P

    2014-01-01

    The research on manipulation of crop genomes for transgenic development is continuously increasing due to several benefits. The major concerns linked to the effect of transgenic crops are human health and environment sustainability. To monitor transgenic samples in the food chain, several highly sensitive and specific DNA-based and protein-based detection methods are being used. However, real- time immunio-PCR (RT-IPCR) assay would be able to provide a sensitive detection of trace amounts of transgenic proteins or allergens in the samples and help in monitoring these materials. In the present study, we developed a novel RT-IPCR method to monitor CrylAc transgenic protein in samples with an LOD of 100 pg/mL. The assay may also be useful in the evaluation of functional stability of transgenes inserted in the plant genome.

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

    Directory of Open Access Journals (Sweden)

    Susan K Morton

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

  20. Characterization of expression of Puumala virus nucleocapsid protein in transgenic plants.

    Science.gov (United States)

    Khattak, Shahryar; Darai, Gholamreza; Süle, Sandor; Rösen-Wolff, Angela

    2002-01-01

    Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 microg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest.

  1. Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates

    NARCIS (Netherlands)

    Souza, M.T.; Níckel, O.; Gonsalves, D.

    2005-01-01

    Line 63-1 is a 'Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R, plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA),

  2. Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates

    NARCIS (Netherlands)

    Souza, M.T.; Níckel, O.; Gonsalves, D.

    2005-01-01

    Line 63-1 is a 'Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R, plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA), Thai

  3. Practical use of corrected fluorescence excitation and emission spectra of fluorescent proteins in Förster Resonance Energy Transfer (FRET) studies

    NARCIS (Netherlands)

    Hink, M.A.; Visser, N.V.; Borst, J.W.; Hoek, van A.; Visser, A.J.W.G.

    2003-01-01

    Corrected fluorescence excitation and emission spectra have been obtained from several enhanced variants of the green fluorescent protein (EGFP) isolated from the jellyfish Aequorea victoria, blue fluorescence protein (EBFP), cyan fluorescent protein (ECFP), EGFP and yellow fluorescent protein (EYFP

  4. Practical use of corrected fluorescence excitation and emission spectra of fluorescent proteins in Förster Resonance Energy Transfer (FRET) studies

    NARCIS (Netherlands)

    Hink, M.A.; Visser, N.V.; Borst, J.W.; Hoek, van A.; Visser, A.J.W.G.

    2003-01-01

    Corrected fluorescence excitation and emission spectra have been obtained from several enhanced variants of the green fluorescent protein (EGFP) isolated from the jellyfish Aequorea victoria, blue fluorescence protein (EBFP), cyan fluorescent protein (ECFP), EGFP and yellow fluorescent protein

  5. Versatile protein tagging in cells with split fluorescent protein

    Science.gov (United States)

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A.; Ishikawa, Hiroaki; Leonetti, Manuel D.; Marshall, Wallace F.; Weissman, Jonathan S.; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  6. Versatile protein tagging in cells with split fluorescent protein.

    Science.gov (United States)

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A; Ishikawa, Hiroaki; Leonetti, Manuel D; Marshall, Wallace F; Weissman, Jonathan S; Huang, Bo

    2016-03-18

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.

  7. Genetically encoded biosensors based on engineered fluorescent proteins.

    Science.gov (United States)

    Frommer, Wolf B; Davidson, Michael W; Campbell, Robert E

    2009-10-01

    Fluorescent proteins have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. While the most common applications of fluorescent proteins are to image expression, localization, and dynamics of protein chimeras, there is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. This tutorial review provides an overview of the progress made in the development of fluorescent protein-based biosensors to date.

  8. Evidence for covalent binding of epicocconone with proteins from synchronous fluorescence spectra and fluorescence lifetimes

    Indian Academy of Sciences (India)

    Debashis Panda; Anindya Datta

    2007-03-01

    Synchronous fluorescence and time-resolved fluorescence spectroscopic studies that reveal the interaction of epicocconone with human serum albumin is significantly different from its interaction with surfactant assemblies. This observation, along with steady-state fluorescence data, indicates groundstate interaction between the fluorophore epicocconone and the protein. Similarity in fluorescence properties with the adduct of the fluorophore with -butylamine indicates that bonding occurs at the Nterminus of the protein.

  9. Photoactivation and imaging of optical highlighter fluorescent proteins.

    Science.gov (United States)

    Patterson, George H

    2011-07-01

    A major advance in the microscopic study of cells and tissues is the introduction of photoactivatable fluorescent proteins, which can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be "turned on" by photoactivation. This unit discusses technical aspects for the general use of photoactivatable fluorescent proteins and introduces some specific applications in the concluding remarks.

  10. Expression of the Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize.

    Science.gov (United States)

    Shou, Huixia; Bordallo, Patricia; Wang, Kan

    2004-05-01

    Drought is one of the most important abiotic stresses affecting the productivity of maize. Previous studies have shown that expression of a mitogen-activated protein kinase kinase kinase (MAPKKK) gene activated an oxidative signal cascade and led to the tolerance of freezing, heat, and salinity stress in transgenic tobacco. To analyse the role of activation of oxidative stress signalling in improving drought tolerance in major crops, a tobacco MAPKKK (NPK1) was expressed constitutively in maize. Results show that NPK1 expression enhanced drought tolerance in transgenic maize. Under drought conditions, transgenic maize plants maintained significantly higher photosynthesis rates than did the non-transgenic control, suggesting that NPK1 induced a mechanism that protected photosynthesis machinery from dehydration damage. In addition, drought-stressed transgenic plants produced kernels with weights similar to those under well-watered conditions, while kernel weights of drought-stressed non-transgenic control plants were significantly reduced when compared with their non-stressed counterparts.

  11. Preparation of fluorescence starch-nanoparticle and its application as plant transgenic vehicle

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Starch-nanoparticles were synthesized in water-in-oil microemusion at room temperature,and the starch-nanoparticles were coated with poly-L-lysine.The surface of the starch-nanoparticles was combined with fluorescence material Ru(bpy)32+·6H2O,and then the particles were characterized via transmission electyron microscope.The fluorescence nanoparticles were conjugasted with plasmid DNA to form complexes,and then treated with ultrasound and Dnase I.pEGAD plasmid DNA-nanoparticle complexes wereco-cultured with plant suspension cells of Dioscrea Zigiberensis G H Wright,and treated with ultrasound.The results show that the diameter of the fluorescence starch-nanoparticles in 50-100nm.DNA-nanoparticle complexes can protect DNA from ultrasound damage as well as from Dnase I cleavage.Mediated by ultrasound,pEGAD plasmid DNA-nanoparticle complexes can pierce into the cell wall,cell membrane and nucleus membrane of plant suspension cells.Thegreen fluorescence protein(GFP)gene at a high frequency exceeds 5%.This nano-biomaterial can efficiently solve the problem that exterior genes cannot traverse the plant cell wall easily.

  12. Non-excitable fluorescent protein orthologs found in ctenophores.

    Science.gov (United States)

    Francis, Warren R; Christianson, Lynne M; Powers, Meghan L; Schnitzler, Christine E; D Haddock, Steven H

    2016-08-24

    Fluorescent proteins are optically active proteins found across many clades in metazoans. A fluorescent protein was recently identified in a ctenophore, but this has been suggested to derive from a cnidarian, raising again the question of origins of this group of proteins. Through analysis of transcriptome data from 30 ctenophores, we identified a member of an orthologous group of proteins similar to fluorescent proteins in each of them, as well as in the genome of Mnemiopsis leidyi. These orthologs lack canonical residues involved in chromophore formation, suggesting another function. The phylogenetic position of the ctenophore protein family among fluorescent proteins suggests that this gene was present in the common ancestor of all ctenophores and that the fluorescent protein previously found in a ctenophore actually derives from a siphonophore.

  13. Ultrafast excited state dynamics of the green fluorescent protein chromophore and its kindling fluorescent protein analogue.

    Science.gov (United States)

    Addison, Kiri; Heisler, Ismael A; Conyard, Jamie; Dixon, Tara; Page, Philip C Bulman; Meech, Stephen R

    2013-01-01

    Fluorescent proteins exhibit a very diverse range of photochemical behaviour, from efficient fluorescence through photochromism to photochemical reactivity. Remarkably this diverse behaviour arises from chromophores which have very similar structures. Here we describe measurements and modelling of the excited state dynamics in the chromophores of GFP (HBDI) and the kindling fluorescent protein, KFP (FHBMI). The methods are ultrafast fluorescence spectroscopy with sub 50 fs time resolution and the modelling is based on the Smoluchowski equation. The excited state decays of both chromophores are very fast, longer for their anions than for the neutral form and independent of wavelength. Detailed studies show the mean fluorescence wavelength to be independent of time. The excited state decay times are also observed to be a very weak function of solvent polarity and viscosity. These results are modelled utilising recently calculated potential energy surfaces for the ground and excited states as a function of the twist coordinates about the two bridging bonds of the chromophore. For FHBMI and the scarce data on the neutral HBDI the calculations are not successful suggesting the need for refinement of these potential energy surfaces. For HBDI in methanol the simulation is successful provided a strong dependence of the radiationless decay rate on the coordinate is assumed. Such dependence should be included in future calculations of excited state dynamics. When the simulations are extended to more viscous solvents they fail to reproduce the observed weak viscosity dependence. The implications of these results for the nature of the coordinate leading to radiationless decay in the chromophore and for the photodynamics of fluorescent proteins are discussed.

  14. Single fluorescent protein-based Ca2+ sensors with increased dynamic range

    Directory of Open Access Journals (Sweden)

    Labas Yulii A

    2007-06-01

    Full Text Available Abstract Background Genetically encoded sensors developed on the basis of green fluorescent protein (GFP-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP. Results Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. Conclusion We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

  15. MYMIV-AC2, a geminiviral RNAi suppressor protein, has potential to increase the transgene expression.

    Science.gov (United States)

    Rahman, Jamilur; Karjee, Sumona; Mukherjee, Sunil Kumar

    2012-06-01

    Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII.

  16. A never ending race for new and improved fluorescent proteins.

    Science.gov (United States)

    Jones, Alexander M; Ehrhardt, David W; Frommer, Wolf B

    2012-05-03

    Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants.

  17. Inhibiting p38 mitogen-activated protein kinase attenuates cerebral ischemic injury in Swedish mutant amyloid precursor protein transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Liangyu Zou; Haiyan Qin; Yitao He; Heming Huang; Yi Lu; Xiaofan Chu

    2012-01-01

    Cerebral ischemia was induced using photothrombosis 1 hour after intraperitoneal injection of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB239063 into Swedish mutant amyloid precursor protein (APP/SWE) transgenic and non-transgenic mice. The number of surviving neurons in the penumbra was quantified using Nissl staining, and the activity of p38 MAPKs was measured by western blotting. The number of surviving neurons in the penumbra was significantly reduced in APP/SWE transgenic mice compared with non-transgenic controls 7 days after cerebral ischemia, but the activity of p38 MAPKs was significantly elevated compared with the non-ischemic hemisphere in the APP/SWE transgenic mice. SB239063 prevented these changes. The APP/SWE mutation exacerbated ischemic brain injury, and this could be alleviated by inhibiting p38 MAPK activity.

  18. Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice

    Directory of Open Access Journals (Sweden)

    Papaioannou Virginia E

    2004-12-01

    Full Text Available Abstract Background Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications. Results We have used embryonic stem cell-mediated transgenesis to label cells at sub-cellular resolution in vivo, and to evaluate fusion of a human histone protein to green fluorescent protein for ubiquitous fluorescent labeling of nucleosomes in mice. To this end we have generated embryonic stem cells and a corresponding strain of mice that is viable and fertile and exhibits widespread chromatin-localized reporter expression. High levels of transgene expression are maintained in a constitutive manner. Viability and fertility of homozygous transgenic animals demonstrates that this reporter is developmentally neutral and does not interfere with mitosis or meiosis. Conclusions Using various optical imaging modalities including wide-field, spinning disc confocal, and laser scanning confocal and multiphoton excitation microscopy, we can identify cells in various stages of the cell cycle. We can identify cells in interphase, cells undergoing mitosis or cell death. We demonstrate that this histone fusion reporter allows the direct visualization of active chromatin in situ. Since this reporter segments three-dimensional space, it permits the visualization of individual cells within a population, and so facilitates tracking cell position over time. It is therefore attractive for use in multidimensional studies of in vivo cell behavior and cell fate.

  19. First evidence of intraclonal genetic exchange in trypanosomatids using two Leishmania infantum fluorescent transgenic clones.

    Directory of Open Access Journals (Sweden)

    Estefanía Calvo-Álvarez

    2014-09-01

    Full Text Available The mode of reproduction in Leishmania spp has been argued to be essentially clonal. However, recent data (genetic analysis of populations and co-infections in sand flies have proposed the existence of a non-obligate sexual cycle in the extracellular stage of the parasite within the sand fly vector. In this article we propose the existence of intraclonal genetic exchange in the natural vector of Leishmania infantum.We have developed transgenic L. infantum lines expressing drug resistance markers linked to green and red fluorescent reporters. We hypothesized whether those cells with identical genotype can recognize each other and mate. Both types of markers were successfully exchanged within the sand fly midgut of the natural vector Phlebotomus perniciosus when individuals from these species were fed with a mixture of parental clones. Using the yellow phenotype and drug resistance markers, we provide evidence for genetic exchange in L. infantum. The hybrid progeny appeared to be triploid based on DNA content analysis. The hybrid clone analyzed was stable throughout the complete parasite life cycle. The progress of infections by the hybrid clone in BALB/c mice caused a reduction in parasite loads in both spleen and liver, and provided weight values similar to those obtained with uninfected mice. Spleen arginase activity was also significantly reduced relative to parental strains.A L. infantum hybrid lineage was obtained from intraclonal genetic exchange within the midgut of the natural vector, suggesting the ability of this parasite to recognize the same genotype and mate. The yellow hybrid progeny is stable throughout the whole parasite life cycle but with a slower virulence, which correlates well with the lower arginase activity detected both in vitro and in vivo infections.

  20. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

    Science.gov (United States)

    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

  1. CHANGES IN ENDOGENOUS GENE TRANSCRIPT AND PROTEIN LEVELS IN MAIZE PLANTS EXPRESSING THE SOYBEAN FERRITIN TRANSGENE

    Directory of Open Access Journals (Sweden)

    Milly N Kanobe

    2013-06-01

    Full Text Available Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337 on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls.

  2. Generation of the regulatory protein rtTA transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Kang Xu; Xin-Yan Deng; Ying Yue; Zhong-Min Guo; Bing Huang; Xun Hong; Dong Xiao; Xi-Gu Chen

    2005-01-01

    AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome "immune tolerance" formed during the embryonic development and "immune escape" against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

  3. Protein conformation in solution by three-dimensional fluorescence spectrometry

    Institute of Scientific and Technical Information of China (English)

    鄢远; 许金钩; 陈国珍

    1996-01-01

    The conformations of bovine serum albumin (USA) and egg albumin (EA) in solution and their conformation changes under different conditions were studied by using three-dimensional fluorescence spectrometry (TDFS) such as three-dimensional fluorescence (TDF) spectra and three-dimensional fluorescence polarization (TDFP) spectra with tryptophan residues in protein molecules as an intrinsic fluorescent probe. The results show that the microenvironment of tryptophan residues of protein molecules in various solutions can be directly indicated and TDFS is an effective tool for studying protein conformation in solution. Meantime, some valuable results were obtained.

  4. Toward fluorescence detection of protein residues on surgical instruments

    Science.gov (United States)

    Richardson, Patricia R.; Jones, Anita C.; Baxter, Robert L.; Baxter, Helen C.; Whittaker, A. Gavin; Campbell, Gaynor A.

    2004-06-01

    Prion proteins are the infectious agents that cause Creutzfeldt-Jakob Disease (CJD) in humans. These proteins are particularly resistant to normal sterilization procedures, and the theoretical risk of prion transmission via surgical instruments is of current public and professional concern. We are currently investigating fluorescence methods for the detection of proteins on surfaces, with a view to developing an optical-fiber-based system for routine, online monitoring of residual protein contamination on surgical instruments, in hospital sterilization departments. This paper presents preliminary results on the detection of femtomole amounts of fluorescently labelled protein on surgical steel and discusses some of the problems involved in the detection of fluorescence from metal samples.

  5. Fluorescence of Alexa Fluor dye tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Borst, J.W.; Visser, A.J.W.G.; Mierlo, van C.P.M.

    2012-01-01

    Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the

  6. Patterns of fluorescent protein expression in Scleractinian corals.

    Science.gov (United States)

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  7. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    Directory of Open Access Journals (Sweden)

    Luchakivska Yu. S.

    2015-08-01

    Full Text Available Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the salinity and osmotic stress tolerance by plant survival test in presence of NaCl and PEG in different concentrations. Results. Transgenic plants able to express recombinant thaumatin II gene (transcription proved for 60–100 % were obtained by agrobacterial transformation. The transgenic carrot plant extracts inhibited the growth of the studied phytopathogenic bacteria strains but exhibited no antifungal activity. Survival level of transgenic plants under the salinity and osmotic stress effect was definitely higher comparing to the untransgenic ones. The analysis of the photosynthetic pigment content in the transgenic carrot plants showed no significant difference of this parameter under salinity stress that may indicate a possible protective activity of the recombinant protein. Conclusions. The obtained in our study transgenic carrot and celery plants able to express the recombinant thaumatin II gene were characterized by antibacterial activity and increased tolerance to salinity and osmotic stress factors.

  8. Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements.

    Directory of Open Access Journals (Sweden)

    Bobin George Abraham

    Full Text Available Fluorescence Resonance Energy Transfer (FRET using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM.

  9. From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants.

    Science.gov (United States)

    Hiwasa-Tanase, Kyoko; Hirai, Tadayoshi; Kato, Kazuhisa; Duhita, Narendra; Ezura, Hiroshi

    2012-03-01

    The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.

  10. In vivo cellular imaging using fluorescent proteins - Methods and Protocols

    Directory of Open Access Journals (Sweden)

    M. Monti

    2012-12-01

    Full Text Available The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible to the cell often can be made visible with the use of fluorescent proteins. With this words, Robert M. Hoffman introduces In vivo Cellular Imaging Using Fluorescent proteins, the eighteen chapters book dedicated to the description of how fluorescence proteins have changed the way to analyze cellular processes in vivo. Modern researches aim to study new and less invasive methods able to follow the behavior of different cell types in different biological contexts: for example, how cancer cells migrate or how they respond to different therapies. Also, in vivo systems can help researchers to better understand animal embryonic development so as how fluorescence proteins may be used to monitor different processes in living organisms at the molecular and cellular level.

  11. Comparative proteomics of milk fat globule membrane proteins from transgenic cloned cattle.

    Directory of Open Access Journals (Sweden)

    Shunchao Sui

    Full Text Available The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC cattle, i.e., expressing recombinant human α-lactalbumin (TC-LA, lactoferrin (TC-LF or lysozyme (TC-LZ in the mammary gland, with those from cloned non-transgenic (C and conventionally bred normal animals (N. We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland.

  12. Iron biofortification and homeostasis in transgenic cassava roots expressing an algal iron assimilatory protein, FEA1

    Directory of Open Access Journals (Sweden)

    Uzoma eIhemere

    2012-09-01

    Full Text Available We have engineered the starchy root crop cassava (Manihot esculenta to express the Chlamydomonas reinhardtii iron assimilatory protein, FEA1, in roots to enhance its nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 gm meal. Significantly, the expression of the FEA1 protein did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of iron mediated by the FEA1 protein. Relative to wild-type plants, FEA1 expressing plants had reduced Fe(III chelate reductase activity and gene expression levels consistent with the more efficient uptake of iron in FEA1 transgenic plants. We also show that genes involved in iron homeostasis in cassava have altered tissue-specific patterns of expression in transgenic plants. Steady state transcript levels of the metal-chelate transporter MeYSL1, and the iron storage proteins, MeFER2 and MeFER6, were elevated in various tissues of FEA1 transgenic plants compared to wild-type plants. These results suggest that these gene products play a role in iron translocation and homeostasis in FEA1 transgenic cassava plants. These results are discussed in terms of enhanced strategies for the iron biofortification of plants.

  13. Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Pedersen, C.; Zimny, J.; Becker, D.

    1997-01-01

    transformant showed a totally different integration pattern. Southern analysis confirmed that the inserted genes were segregating independently, resulting in different integration patterns among the progeny lines. The application of the FISH technique for the analysis of transgenic plants is discussed.......Using fluorescence in situ hybridization (FISH) we localized introduced genes on metaphase chromosomes of barley, wheat, and triticale transformed by microprojectile bombardment of microspores and scutellar tissue with the pDB1 plasmid containing the uidA and bar genes. Thirteen integration sites...... of single-copy integrations. There was a slight tendency towards the localization of transgenes in distal chromosome regions. Using the GAA-satellite sequence for chromosome banding, the chromosomes containing the inserted genes were identified in most cases. Two barley lines derived from the same...

  14. Reduction of lesion growth rate of late blight plant disease in transgenic potato expressing harpin protein

    Institute of Scientific and Technical Information of China (English)

    李汝刚; 范云六

    1999-01-01

    Using harpin protein gene from apple fire blight pathogen Erwinia amylavora and potato prp1-1 promoter as main DNA elements, the feasibility of using pathogen infection-induced hypersensitive response was explored as a new strategy of engineering fungal disease resistance. Three plant transformation vectors were constructed and 68 transgenic potato plants were produced through Agrobacterium mediated transformation method. Southern, Northern and Western blot analysis demonstrated the insertion, transcription and protein expression of harpin protein gene in transgenic plants. Disease resistance test using a complex race of Phytophthora infestans as challenging pathogen showed that both constitutive and pathogen infection-induced expression of harpin protein gene in transgenic potato reduced the lesion growth rate of fungus. Among plants where harpin protein gene expression was induced only by fungus infection, two plants were found to be highly resistant to P. infestans infection. Fungal hyphae were not pr

  15. Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins.

    Science.gov (United States)

    François, Isabelle E J A; De Bolle, Miguel F C; Dwyer, Geoff; Goderis, Inge J W M; Woutors, Piet F J; Verhaert, Peter D; Proost, Paul; Schaaper, Wim M M; Cammue, Bruno P A; Broekaert, Willem F

    2002-04-01

    We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.

  16. Ratiometric fluorescent pH nanoprobes based on in situ assembling of fluorescence resonance energy transfer between fluorescent proteins.

    Science.gov (United States)

    Yu, Haijun; Chen, Chao; Cao, Xiaodan; Liu, Yueling; Zhou, Shengmin; Wang, Ping

    2017-08-01

    pH-dependent protein adsorption on mesoporous silica nanoparticle (MSN) was examined as a unique means for pH monitoring. Assuming that the degree of protein adsorption determines the distance separating protein molecules, we examined the feasibility of nanoscale pH probes based on fluorescence resonance energy transfer (FRET) between two fluorescent proteins (mTurquoise2 and mNeonGreen, as donor and acceptor, respectively). Since protein adsorption on MSN is pH-sensitive, both fluorescent proteins were modified to make their isoelectric points (pIs) identical, thus achieving comparable adsorption between the proteins and enhancing FRET signals. The adsorption behaviors of such modified fluorescent proteins were examined along with ratiometric FRET signal generation. Results demonstrated that the pH probes could be manipulated to show feasible sensitivity and selectivity for pH changes in hosting solutions, with a good linearity observed in the pH range of 5.5-8.0. In a demonstration test, the pH probes were successfully applied to monitor progress of enzymatic reactions. Such an "in situ-assembling" pH sensor demonstrates a promising strategy in developing nanoscale fluorescent protein probes. Graphical abstract Working principle of the developed pH sensor TNS; and FRET Ratio (I528/I460) as a function of pH under different protein feed ratios (mNeonGreen to mTurquoise2).

  17. An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein

    Science.gov (United States)

    Baker, Stokes S.; Vidican, Cleo B.; Cameron, David S.; Greib, Haittam G.; Jarocki, Christine C.; Setaputri, Andres W.; Spicuzza, Christopher H.; Burr, Aaron A.; Waqas, Meriam A.; Tolbert, Danzell A.

    2012-01-01

    Background and aims Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants. Methodology Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically. Principal findings Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H0: β (slope) = 0 mean counts per pixel (ng s mm−2)−1, r2 > 0.994, n = 31, P < 1.75 × 10−29). Conclusions Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with

  18. Recombination with coat protein transgene in a complemen-tation system based on Cucumber mosaic virus (CMV)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an NsiⅠ site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants ex-pressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination between the CMV vector and the CP transgene was proved by retro-transcriptional polymerase chain reaction (RT-PCR) and verified by DNA sequencing. Our results argue against the feasibility of the CMV-based replace-ment vector trans-complemented by the CP transgene, and at the same time, enlighten ways to improve the CMV-based expression vector and the biosafety of CMV CP-mediated virus resistant transgenic plants.

  19. Quenching of fluorescence in membrane protein by hypocrellin B.

    Science.gov (United States)

    Yue, J; Pang, S

    1997-04-01

    The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical charactcristics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quenchtr between membrane and water, and the fluorescence quenching constant of protein (K(sv); K(q),). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was observed in detail by using the ESR technique. The signal of HB- was found to arise from an electron transfer from excited trytophan to HB.

  20. Quenching of fluorescence in membrane protein by hypocrellin B

    Institute of Scientific and Technical Information of China (English)

    乐加昌; 庞素珍

    1997-01-01

    The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical characteris-tics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quencher between membrane and water, and the fluorescence quenching constant of protein (Ksv; Kq). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was ob-served in detail by using the ESR technique. The signal of HB" was found to arise from an electron transfer from ex-cited trytophan to HB.

  1. Fluorescence of Alexa fluor dye tracks protein folding.

    Science.gov (United States)

    Lindhoud, Simon; Westphal, Adrie H; Visser, Antonie J W G; Borst, Jan Willem; van Mierlo, Carlo P M

    2012-01-01

    Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.

  2. Fluorescence of Alexa fluor dye tracks protein folding.

    Directory of Open Access Journals (Sweden)

    Simon Lindhoud

    Full Text Available Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488, which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.

  3. Transgenic bioreactors.

    Science.gov (United States)

    Jänne, J; Alhonen, L; Hyttinen, J M; Peura, T; Tolvanen, M; Korhonen, V P

    1998-01-01

    Since the generation of the first transgenic mice in 1980, transgene technology has also been successfully applied to large farm animals. Although this technology can be employed to improve certain production traits of livestock, this approach has not been very successful so far owing to unwanted effects encountered in the production animals. However, by using tissue-specific targeting of the transgene expression, it is possible to produce heterologous proteins in the extracellular space of large transgenic farm animals. Even though some recombinant proteins, such as human hemoglobin, have been produced in the blood of transgenic pigs, in the majority of the cases mammary gland targeted expression of the transgene has been employed. Using production genes driven by regulatory sequences of milk protein genes a number of valuable therapeutic proteins have been produced in the milk of transgenic bioreactors, ranging from rabbits to dairy cattle. Unlike bacterial fermentors, the mammary gland of transgenic bioreactors appear to carry out proper postsynthetic modifications of human proteins required for full biological activity. In comparison with mammalian cell bioreactors, transgenic livestock with mammary gland targeted expression seems to be able to produce valuable human therapeutic proteins at very low cost. Although not one transgenically produced therapeutic protein is yet on the market, the first such proteins have recently entered or even completed clinical trials required for their approval.

  4. Fluorescence Quantum Yield Measurements of Fluorescent Proteins: A Laboratory Experiment for a Biochemistry or Molecular Biophysics Laboratory Course

    Science.gov (United States)

    Wall, Kathryn P.; Dillon, Rebecca; Knowles, Michelle K.

    2015-01-01

    Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts…

  5. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; Berg, van den Timo K.; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase

  6. Production of a Transgenic Mosquito Expressing Circumsporozoite Protein, a Malarial Protein, in the Salivary Gland of Anopheles stephensi (Diptera:Culicidae

    Directory of Open Access Journals (Sweden)

    Matsuoka,Hiroyuki

    2010-08-01

    Full Text Available We are producing a transgenic mosquito, a flying syringe, to deliver a vaccine protein to human beings via the saliva the mosquito deposits in the skin while biting. The mosquito produces a vaccine protein in the salivary gland (SG and deposits the protein into the host's skin when it takes the host's blood. We chose circumsporozoite protein (CSP, currently the most promising malaria vaccine candidate, to be expressed in the SG of Anopheles stephensi. To transform the mosquitoes, plasmid containing the CSP gene under the promoter of female SG-specific gene, as well as the green fluorescent protein (GFP gene under the promoter of 3xP3 as a selection marker in the eyes, was injected into more than 400 eggs. As a result, five strains of GFP-expressing mosquitoes were established, and successful CSP expression in the SG was confirmed in one strain. The estimated amount of CSP in the SG of the strain was 40ng per mosquito. We allowed the CSP-expressing mosquitoes to feed on mice to induce the production of anti-CSP antibody. However, the mice did not develop anti-CSP antibody even after transgenic mosquitoes had bitten them several times. We consider that CSP in the SG was not secreted properly into the saliva. Further techniques and trials are required in order to realize vaccine-delivering mosquitoes.

  7. A never ending race for new and improved fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Jones Alexander M

    2012-05-01

    Full Text Available Abstract Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants. See research article http://www.biomedcentral.com/1472-6750/12/17

  8. A never ending race for new and improved fluorescent proteins

    Science.gov (United States)

    2012-01-01

    Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants. See research article http://www.biomedcentral.com/1472-6750/12/17 PMID:22554191

  9. Common fluorescent proteins for single-molecule localization microscopy

    Science.gov (United States)

    Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-07-01

    Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

  10. Establishment of transgenic mice carrying gene encoding human zinc finger protein 191

    Science.gov (United States)

    Li, Jian-Zhong; Chen, Xia; Yang, Hua; Wang, Shui-Liang; Gong, Xue-Lian; Feng, Hao; Guo, Bao-Yu; Yu, Long; Wang, Zhu-Gang; Fu, Ji-Liang

    2004-01-01

    AIM: Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Krüppel-like transcription factor, which might be relevant to many diseases such as liver cancer, neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF 191 transgenic mouse model, which would promote the functional study of ZNF191. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination. RESULTS: Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF 191 gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF 191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice. CONCLUSION: ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo. PMID:14716836

  11. A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

    Science.gov (United States)

    Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

    2015-01-01

    Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

  12. A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

    Science.gov (United States)

    Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

    2015-01-01

    Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

  13. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET

    NARCIS (Netherlands)

    Subach, F.V.; Zhang, L.; Gadella, T.W.J.; Gurskaya, N.G.; Lukyanov, K.A.; Verkhusha, V.V.

    2010-01-01

    We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF stat

  14. Fluorescence labeling of carbon nanotubes and visualization of a nanotube-protein hybrid under fluorescence microscope.

    Science.gov (United States)

    Yoshimura, Shige H; Khan, Shahbaz; Maruyama, Hiroyuki; Nakayama, Yoshikazu; Takeyasu, Kunio

    2011-04-11

    Biological applications of carbon nanotubes have been hampered by the inability to visualize them using conventional optical microscope, which is the most common tool for the observation and measurement of biological processes. Recently, a number of fluorescence labeling methods for biomolecules and various fluorescence probes have been developed and widely utilized in biological fields. Therefore, labeling carbon nanotubes with such fluorophores under physiological conditions will be highly useful in their biological applications. In this Article, we present a method to fluorescently label nanotubes by combining a detergent and a fluorophore commonly used in biological experiments. Fluorophores carrying an amino group (Texas Red hydrazide or BODIPY FL-hydrazide) were covalently attached to the hydroxyl groups of Tween 20 using carbonyldiimidazole. Fluorescence microscopy demonstrated that nanotubes were efficiently solubilized and labeled by this fluorescently labeled detergent. By using this technique, we also demonstrated multicolor fluorescence imaging of a nanotube-protein hybrid.

  15. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

    Directory of Open Access Journals (Sweden)

    Mudrik Nikolay N

    2002-04-01

    Full Text Available Abstract Background Within the family of green fluorescent protein (GFP homologs, one can mark two main groups, specifically, fluorescent proteins (FPs and non-fluorescent or chromoproteins (CPs. Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595 from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield ( Conclusions We located a novel point in asCP sequence (position 165 mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

  16. Influence of Phytase Transgenic Corn on the Intestinal Microflora and the Fate of Transgenic DNA and Protein in Digesta and Tissues of Broilers.

    Science.gov (United States)

    Lu, Lin; Guo, Jiang; Li, Sufen; Li, Ang; Zhang, Liyang; Liu, Zhenhua; Luo, Xugang

    2015-01-01

    An experiment was conducted to investigate the effect of phytase transgenic corn (PTC) on intestinal microflora, and the fate of transgenic DNA and protein in the digesta and tissues of broilers. A total of 160 1-day-old Arbor Acres commercial male broilers were randomly assigned to 20 cages (8 chicks per cage) with 10 cages (replicates) for each treatment. Birds were fed with a diet containing either PTC (54.0% during 1-21 days and 61.0% during 22-42 days) or non-transgenic isogenic control corn (CC) for a duration of 42 days. There were no significant differences (P>0.05) between birds fed with the PTC diets and those fed with the CC diets in the quantities of aerobic bacteria, anaerobic bacteria, colibacillus and lactobacilli, or microbial diversities in the contents of ileum and cecum. Transgenic phyA2 DNA was not detected, but phyA2 protein was detected in the digesta of duodenum and jejunum of broilers fed with the PTC diets. Both transgenic phyA2 DNA and protein fragments were not found in the digesta of the ileum and rectum, heart, liver, kidney, and breast or thigh muscles of broilers fed with the PTC diets. It was concluded that PTC had no adverse effect on the quantity and diversity of gut microorganisms; Transgenic phyA2 DNA or protein was rapidly degraded in the intestinal tract and was not transferred to the tissues of broilers.

  17. Influence of Phytase Transgenic Corn on the Intestinal Microflora and the Fate of Transgenic DNA and Protein in Digesta and Tissues of Broilers.

    Directory of Open Access Journals (Sweden)

    Lin Lu

    Full Text Available An experiment was conducted to investigate the effect of phytase transgenic corn (PTC on intestinal microflora, and the fate of transgenic DNA and protein in the digesta and tissues of broilers. A total of 160 1-day-old Arbor Acres commercial male broilers were randomly assigned to 20 cages (8 chicks per cage with 10 cages (replicates for each treatment. Birds were fed with a diet containing either PTC (54.0% during 1-21 days and 61.0% during 22-42 days or non-transgenic isogenic control corn (CC for a duration of 42 days. There were no significant differences (P>0.05 between birds fed with the PTC diets and those fed with the CC diets in the quantities of aerobic bacteria, anaerobic bacteria, colibacillus and lactobacilli, or microbial diversities in the contents of ileum and cecum. Transgenic phyA2 DNA was not detected, but phyA2 protein was detected in the digesta of duodenum and jejunum of broilers fed with the PTC diets. Both transgenic phyA2 DNA and protein fragments were not found in the digesta of the ileum and rectum, heart, liver, kidney, and breast or thigh muscles of broilers fed with the PTC diets. It was concluded that PTC had no adverse effect on the quantity and diversity of gut microorganisms; Transgenic phyA2 DNA or protein was rapidly degraded in the intestinal tract and was not transferred to the tissues of broilers.

  18. High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus dragline silk protein.

    Directory of Open Access Journals (Sweden)

    Yoshihiko Kuwana

    Full Text Available Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol% native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

  19. High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein.

    Science.gov (United States)

    Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

    2014-01-01

    Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

  20. Transgenic biofortification of the starchy staple cassava (Manihot esculenta) generates a novel sink for protein.

    Science.gov (United States)

    Abhary, Mohammad; Siritunga, Dimuth; Stevens, Gene; Taylor, Nigel J; Fauquet, Claude M

    2011-01-25

    Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz). (Montagnac JA, Davis CR, Tanumihardjo SA. (2009) Compr Rev Food Sci Food Saf 8:181-194). Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications.

  1. Fluorescence properties of porcine odorant binding protein Trp 16 residue

    Energy Technology Data Exchange (ETDEWEB)

    Albani, Jihad Rene, E-mail: Jihad-Rene.Albani@univ-lille1.f [Laboratoire de Biophysique Moleculaire, Universite des Sciences et Technologies de Lille, F-59655 Villeneuve d' Ascq Cedex (France)

    2010-11-15

    Summary: The present work deals with fluorescence studies of adult porcine odorant binding protein at pH=7.5. At this pH, the protein is a dimer, each monomer contains one tryptophan residue. Our results show that tryptophan residue displays significant motions and emits with three fluorescence lifetimes. Decay associated spectra showed that the three lifetime's components emanate from sub-structures surrounded by the same microenvironment.

  2. Origin of tryptophan fluorescence lifetimes. Part 2: fluorescence lifetimes origin of tryptophan in proteins.

    Science.gov (United States)

    Albani, J R

    2014-01-01

    Fluorescence intensity decays of L-tryptophan in proteins dissolved in pH 7 buffer, in ethanol and in 6 M guanidine pH 7.8 and in lyophilized proteins were measured. In all protein conditions, three lifetimes were obtained along the emission spectrum (310-410 nm). The two shortest lifetimes are in the same range of those obtained for L-Trp in water or in ethanol. Thus, these two lifetimes originate from specific two sub-structures existing in the excited state and are inherent to the tryptophan structure independently of the surrounding environment (amino acids residues, solvent, etc.) In proteins, the third lifetime originates from the interactions that are occurring between tryptophan residues and neighboring amino acids. Populations of these lifetimes are independent of the excitation wavelength and thus originate from pre-defined sub structures existing in the excited state and put into evidence after tryptophan excitation. Fluorescence decay studies of different tripeptides having a tryptophan residue in second position show that the best analysis is obtained with two fluorescence lifetimes. Consequently, this result seems to exclude the possibility that peptide bond induces the third fluorescence lifetimes. Indole dissolved in water and/or in ethanol emits with two fluorescence lifetimes that are completely different from those observed for L-Trp. Absence of the third lifetime in ethanol demonstrates that indole behaves differently when compared to tryptophan. Thus, it seems not adequate to attribute fluorescence lifetime or fluorescence properties of tryptophan to indole ring and to compare tryptophan fluorescence properties in proteins to molecules having close structures such as NATA which fluoresces with one lifetime.

  3. In vivo cell biology of cancer cells visualized with fluorescent proteins.

    Science.gov (United States)

    Hoffman, Robert M

    2005-01-01

    This chapter describes a new cell biology where the behavior of individual cells can be visualized in the living animal. Previously it has been demonstrated that fluorescent proteins can be used for whole-body imaging of metastatic tumor growth, bacterial infection, and gene expression. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of tumor-stroma interactions and especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts, and macrophages. Another example is the color coding of cells with RFP or GFP such that both cell types can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo. Mice in which the regulatory elements of the stem cell marker nestin drive GFP expression enable nascent vasculature to be visualized interacting with transplanted RFP-expressing cancer cells. Nestin-driven GFP expression can also be used to visualize hair follicle stem cells. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Highly elongated cancer cells in capillaries in living mice were observed within skin flaps. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells in the capillaries elongated to fit the width of these vessels. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

  4. Construction and Quality Analysis of Transgenic Rehmannia glutinosa Containing TMV and CMV Coat Protein

    Directory of Open Access Journals (Sweden)

    Zhongqiu Teng

    2016-08-01

    Full Text Available Plant viruses, especially tobacco mosaic virus (TMV and cucumber mosaic virus (CMV are serious threats to Rehmannia glutinosa which is a “top grade” herb in China. In the present study, TMV- and CMV-resistant Rehmannia glutinosa Libosch. plants were constructed by transforming the protein (CP genes of TMV and CMV into Rehmannia glutinosa via a modified procedure of Agrobacterium tumefaciens-mediated transformation. Integration and expression of TMV CP and CMV CP transgenes in 2 lines, LBA-1 and LBA-2, were confirmed by PCR, Southern blot and RT-PCR. Both LBA-1 and LBA-2 were resistant to infection of homologous TMV and CMV strains. The quality of transgenic Rehmanniae Radix was evaluated based on fingerprint analysis and components quantitative analysis comparing with control root tubes. These results showed that chemical composition of transgenic Rehmanniae Radix were similar to non-transgenic ones, which demonstrated that the medical quality and biosafety of transgenic Rehmanniae Radix were equivalent to non-transgenic material when consumed as traditional Chinese medicinal (TCM.

  5. Construction and Quality Analysis of Transgenic Rehmannia glutinosa Containing TMV and CMV Coat Protein.

    Science.gov (United States)

    Teng, Zhongqiu; Shen, Ye; Li, Jing; Lin, Zhongping; Chen, Min; Wang, Min; Li, Man; Dong, Hongran; Huang, Luqi

    2016-08-27

    Plant viruses, especially tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are serious threats to Rehmannia glutinosa which is a "top grade" herb in China. In the present study, TMV- and CMV-resistant Rehmannia glutinosa Libosch. plants were constructed by transforming the protein (CP) genes of TMV and CMV into Rehmannia glutinosa via a modified procedure of Agrobacterium tumefaciens-mediated transformation. Integration and expression of TMV CP and CMV CP transgenes in 2 lines, LBA-1 and LBA-2, were confirmed by PCR, Southern blot and RT-PCR. Both LBA-1 and LBA-2 were resistant to infection of homologous TMV and CMV strains. The quality of transgenic Rehmanniae Radix was evaluated based on fingerprint analysis and components quantitative analysis comparing with control root tubes. These results showed that chemical composition of transgenic Rehmanniae Radix were similar to non-transgenic ones, which demonstrated that the medical quality and biosafety of transgenic Rehmanniae Radix were equivalent to non-transgenic material when consumed as traditional Chinese medicinal (TCM).

  6. A Standardized Lepidopteran Bioassay to Investigate the Bioactivity of Insecticidal Proteins Produced in Transgenic Crops.

    Science.gov (United States)

    Graser, Gerson; Walters, Frederick S

    2016-01-01

    Insecticidal bioassays are the only reliable method to investigate the biological activity of an insecticidal protein and therefore provide an essential toolkit for the characterization and potency determination of these proteins. Here we present a standardized method for a lepidopteran larval bioassay, which is optimized to specifically estimate activity of insecticidal proteins produced in transgenic plants. The treatment can be either applied to the surface of the artificial diet, or blended into the diet.

  7. High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.

    OpenAIRE

    Wall, R J; Pursel, V G; Shamay, A; McKnight, R A; Pittius, C W; Hennighausen, L

    1991-01-01

    The whey acidic protein (WAP) is a major milk protein in mice, rats, and rabbits but has not been found in milk of livestock including swine. To determine whether mammary gland regulatory elements from the WAP gene function across species boundaries and whether it is possible to qualitatively alter milk protein composition, we introduced the mouse WAP gene into the genome of swine. Three lines of transgenic swine were analyzed, and mouse WAP was detected in milk from all lactating females at ...

  8. Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.

    Science.gov (United States)

    Allen, Aron; Islamovic, Emir; Kaur, Jagdeep; Gold, Scott; Shah, Dilip; Smith, Thomas J

    2011-10-01

    The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.

  9. Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

    NARCIS (Netherlands)

    Bader, A.N.; Visser, N.V.; Amerongen, van H.; Visser, A.J.W.G.

    2014-01-01

    The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and ex

  10. 荧光蛋白在植物活细胞液泡成像中的应用研究进展%Recent Progress in Living Cell Imaging of Plant Vacuole Using Fluorescent-protein Transgenic Lines and Three-dimensional Imaging

    Institute of Scientific and Technical Information of China (English)

    叶庆亮; 曹建华

    2011-01-01

    高等植物细胞中,液泡在各种细胞信号转导事件和形态建成中起着重要的作用.这一细胞内部结构在分裂和分化期间其功能和形状不断地变化着.为分析这些连续的变化,绿色荧光蛋白(green fluorescent protein,GFP)及其他荧光蛋白活体标记技术普遍用来跟踪特异蛋白的定位及变动.为使液泡可视成像,有几种途径可用来选择适当的荧光蛋白融合伴侣,如液泡膜内在蛋白和构造蛋白相关蛋白等就非常适合应用于液泡成像.此外,三维重建法在定位细胞内广为分布的细胞器时也不可或缺.同时,等值面表面模化过程对液泡膜成像也非常有用.本文综述液泡的结构、种类和功能,并概括各种融合的绿色荧光蛋白在植物活细胞液泡成像中的应用及其三维成像技术的研究进展.%In high plant cells, vacuoles play important roles in a variety of cellular events, including cell division,morphogenesis, and signal transduction.These intracellular structures undergo dynamic changes in their shapes and functions during cell division and differentiation, and to analyze these sequential structural changes, the vital labeling technique, using the green-fluorescent protein or other fluorescent proteins, has commonly been used to follow the localization and translocation of specific proteins.To visualize vacuoles, the tonoplast-intrinsic proteins and syntaxin-related proteins are available for selecting the appropriate fluorescent-protein fusion partner for vacuolar imaging.In addition, three-dimensional reconstruction methods are indispensable for localizing the widely distributed organelles within the cell.The maximum intensity projection method is suitable for cytoskeletal structures, while contour-based surface modeling possesses many advantages for vacuolar membranes.In this article, we summarize the plant vacuoles and the recent progress in living cell imaging of the plant vacuoles using various fusions

  11. [Fluorescent fusion proteins with 10th human fibronectin domain].

    Science.gov (United States)

    Petrovskaia, L E; Gapizov, S Sh; Shingarova, L N; Kriukova, E A; Boldyreva, E F; Iakimov, S A; Svirshchevskaia, E V; Lukashev, E P; Dolgikh, D A; Kirpichnikov, M P

    2014-01-01

    In the current paper we describe a new type of hybrid molecules including red fluorescent protein mCherry and 10th type III human fibronectin domain (10Fn3) - one of the alternative scaffold proteins which can be used for the construction of antibody mimics with various binding specificity. We have constructed different gene variants encoding for the hybrid fluorescent protein and studied their expression in Escherichia coli cells. It was shown that N-terminal position of mCherry and modification of its N-terminal amino acid sequence promotes efficientbacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construction we have obtained the hybrid fluorescent protein ChIBF, containing alphaVbeta3-integrin binding vari- ant of 10Fn3, and demonstrated the possibility of its utilization for the visualization of alphaVbeta3-integrin at the surface of MDCK epithelial cells by confocal microscopy.

  12. Islet neogenesis associated protein transgenic mice are resistant to hyperglycemia induced by streptozotocin.

    Science.gov (United States)

    Taylor-Fishwick, David A; Bowman, Angela; Hamblet, Natasha; Bernard, Paul; Harlan, David M; Vinik, Aaron I

    2006-09-01

    Islet neogenesis associated protein (INGAP) is a protein factor that can stimulate new islet mass from adult pancreatic progenitor cells. In models of islet neogenesis, INGAP expression is elevated in pancreatic acinar cells. Using a transgenic model to drive a sustained expression of INGAP in pancreatic acinar cells, we have identified a protection to chemical-induced hyperglycemia. A sustained expression of INGAP during development did not perturb islet development or basal blood glucose homeostasis, although beta-cell mass and pancreatic insulin content were significantly increased in the INGAP transgenic mice. When challenged with a diabetogenic dose of streptozotocin (STZ), mice carrying the INGAP transgene did not become hyperglycemic. In contrast, wild-type mice became and remained hyperglycemic, blood glucose > 550 mg/dl. The serum insulin levels and islet morphology were preserved in the transgenic mice after STZ treatment. These data suggest that the sustained expression of INGAP in the acinar pancreas confers resistance to a diabetogenic insult. The INGAP transgenic mouse provides a new model to uncover factors that are protective to diabetes onset and biomarkers to track beta-cell pathology.

  13. Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

    Directory of Open Access Journals (Sweden)

    Bryan Sands

    Full Text Available Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

  14. Effect of refractive index on the fluorescence lifetime of green fluorescent protein.

    Science.gov (United States)

    Tregidgo, Carolyn; Levitt, James A; Suhling, Klaus

    2008-01-01

    The average fluorescence lifetime of the green fluorescent protein (GFP) in solution is a function of the refractive index of its environment. We report that this is also the case for GFP-tagged proteins in cells. Using time-correlated single-photon counting (TCSPC)-based fluorescence lifetime imaging (FLIM) with a confocal scanning microscope, images of GFP-tagged proteins in cells suspended in different refractive index media are obtained. It is found that the average fluorescence lifetime of GFP decreases on addition of glycerol or sucrose to the media in which the fixed cells are suspended. The inverse GFP lifetime is proportional to the refractive index squared. This is the case for GFP-tagged major histocompatibility complex (MHC) proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP that is located outside the cell membrane. The implications of these findings are discussed with regard to total internal reflection fluorescence (TIRF) techniques where the change in refractive index is crucial in producing an evanescent wave to excite fluorophores near a glass interface. Our findings show that the GFP fluorescence lifetime is shortened in TIRF microscopy in comparison to confocal microscopy.

  15. On the purported "backbone fluorescence" in protein three-dimensional fluorescence spectra

    DEFF Research Database (Denmark)

    Bortolotti, Annalisa; Wong, Yin How; Korsholm, Stine S.

    2016-01-01

    as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence......In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220-230 nm. In the last 10 years we have observed a worrying increase in the number of articles...... claiming that this fluorescence originates from the protein backbone, contrary to the established knowledge that UV protein emission is due to aromatic amino acids only. Overall, our data clearly demonstrate that the observed emission upon excitation at 220-230 nm is due to the excitation of Tyr and/or Trp...

  16. Fluorescence quantum yield measurements of fluorescent proteins: a laboratory experiment for a biochemistry or molecular biophysics laboratory course.

    Science.gov (United States)

    Wall, Kathryn P; Dillon, Rebecca; Knowles, Michelle K

    2015-01-01

    Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum.

  17. Rational design of enhanced photoresistance in a photoswitchable fluorescent protein

    Science.gov (United States)

    Duan, Chenxi; Byrdin, Martin; El Khatib, Mariam; Henry, Xavier; Adam, Virgile; Bourgeois, Dominique

    2015-03-01

    Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions.

  18. Effects of peripherally administered cholecystokinin-8 and secretin on feeding/drinking and oxytocin-mRFP1 fluorescence in transgenic rats.

    Science.gov (United States)

    Motojima, Yasuhito; Kawasaki, Makoto; Matsuura, Takanori; Saito, Reiko; Yoshimura, Mitsuhiro; Hashimoto, Hirofumi; Ueno, Hiromichi; Maruyama, Takashi; Suzuki, Hitoshi; Ohnishi, Hideo; Sakai, Akinori; Ueta, Yoichi

    2016-08-01

    Peripheral administration of cholecystokinin (CCK)-8 or secretin activates oxytocin (OXT)-secreting neurons in the hypothalamus. Although OXT is involved in the regulation of feeding behavior, detailed mechanism remains unclear. In the present study, we examined the central OXTergic pathways after intraperitoneally (i.p.) administration of CCK-8 and secretin using male OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats and male Wistar rats. I.p. administration of CCK-8 (50μg/kg) and secretin (100μg/kg) decreased food intake in these rats. While i.p. administration of CCK-8 decreased water intake, i.p. administration of secretin increased water intake. Immunohistochemical study revealed that Fos-Like-Immunoreactive cells were observed abundantly in the brainstem and in the OXT neurons in the dorsal division of the parvocellular paraventricular nucleus (dpPVN). We could observe marked increase of mRFP1 fluorescence, as an indicator for OXT, in the dpPVN and mRFP1-positive granules in axon terminals of the dpPVN OXT neurons in the nucleus tractus solitarius (NTS) after i.p. administration of CCK-8 and secretin. These results provide us the evidence that, at least in part, i.p. administration of CCK-8 or secretin might be involved in the regulation of feeding/drinking via a OXTergic pathway from the dpPVN to the NTS.

  19. Highly Selective Fluorescent Sensing of Proteins Based on a Fluorescent Molecularly Imprinted Nanosensor

    Directory of Open Access Journals (Sweden)

    Shuo Wang

    2013-09-01

    Full Text Available A fluorescent molecularly imprinted nanosensor was obtained by grafting imprinted polymer onto the surface of multi-wall carbon nanotubes and post-imprinting treatment with fluorescein isothiocyanate (FITC. The fluorescence of lysozyme-imprinted polymer (Lys-MIP was quenched more strongly by Lys than that of nonimprinted polymer (NIP, which indicated that the Lys-MIP could recognize Lys. The resulted imprinted material has the ability to selectively sense a target protein, and an imprinting factor of 3.34 was achieved. The Lys-MIP also showed selective detection for Lys among other proteins such as cytochrome C (Cyt C, hemoglobin (HB and bovine serum albumin (BSA due to the imprinted sites in the Lys-MIP. This approach combines the high selectivity of surface molecular imprinting technology and fluorescence, and converts binding events into detectable signals by monitoring fluorescence spectra. Therefore, it will have further applications for Lys sensing.

  20. Fluorescence resonance energy transfer between fluorescent proteins as powerful toolkits for in vivo studies

    Science.gov (United States)

    Rusanov, A. L.; Savitsky, A. P.

    2011-02-01

    To expand the field of research in biological systems development of extra-sensitive analytical methods is highly desirable. In this review, the latest advances in technologies relying on the fluorescence resonance energy transfer between fluorescent proteins (FP's) to visualize numerous molecular processes in living cells are discussed. Variety of FP's as well as of novel experimental techniques allows one to choose the most appropriate tools to attack concrete problems.

  1. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins.

    OpenAIRE

    2000-01-01

    We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotr...

  2. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    Science.gov (United States)

    Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

    2014-09-01

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

  3. Fluobodies : green fluorescent single-chain Fv fusion proteins

    NARCIS (Netherlands)

    Griep, R.A.; Twisk, van C.; Wolf, van der J.M.; Schots, A.

    1999-01-01

    An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selec

  4. mKikGR, a monomeric photoswitchable fluorescent protein.

    Directory of Open Access Journals (Sweden)

    Satoshi Habuchi

    Full Text Available The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

  5. Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

    Science.gov (United States)

    Permyakova, Natalia V; Zagorskaya, Alla A; Belavin, Pavel A; Uvarova, Elena A; Nosareva, Olesya V; Nesterov, Andrey E; Novikovskaya, Anna A; Zav'yalov, Evgeniy L; Moshkin, Mikhail P; Deineko, Elena V

    2015-01-01

    Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  6. Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce.

    Science.gov (United States)

    Sun, Hyeon-Jin; Cui, Min-Long; Ma, Biao; Ezura, Hiroshi

    2006-01-23

    Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein.

  7. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    Directory of Open Access Journals (Sweden)

    Natalia V. Permyakova

    2015-01-01

    Full Text Available Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L. genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  8. Iron biofortification and homeostasis in transgenic cassava roots expressing an algal iron assimilatory protein, FEA1

    OpenAIRE

    2012-01-01

    We have engineered the starchy root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory protein, FEA1, in roots to enhance its nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 gm meal. Significantly, the expression of the FEA1 protein did not alter iron levels in l...

  9. Spatial Distribution of Transgenic Protein After Gene Electrotransfer to Porcine Muscle

    DEFF Research Database (Denmark)

    Spanggaard, Iben; Corydon, Thomas; Hojman, Pernille

    2012-01-01

    Abstract Gene electrotransfer is an effective nonviral technique for delivery of plasmid DNA into tissues. From a clinical perspective, muscle is an attractive target tissue as long-term, high-level transgenic expression can be achieved. Spatial distribution of the transgenic protein following gene...... electrotransfer to muscle in a large animal model has not yet been investigated. In this study, 17 different doses of plasmid DNA (1-1500 μg firefly luciferase pCMV-Luc) were delivered in vivo to porcine gluteal muscle using electroporation. Forty-eight hours post treatment several biopsies were obtained from...... each transfection site in order to examine the spatial distribution of the transgenic product. We found a significantly higher luciferase activity in biopsies from the center of the transfection site compared to biopsies taken adjacent to the center, 1 and 2 cm along muscle fiber orientation (p...

  10. Production and immunogenicity of Actinobacillus pleuropneumoniae ApxIIA protein in transgenic rice callus.

    Science.gov (United States)

    Kim, Mi-Young; Kim, Tae-Geum; Yang, Moon-Sik

    2017-04-01

    Actinobacillus pleuropneumoniae is a major etiological agent that is responsible for swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA is one of the virulence factors in A. pleuropneumoniae and has been considered as a candidate for developing a vaccine against the bacterial infection. A gene encoding an ApxIIA fragment (amino acids 439-801) was modified based on a plant-optimized codon and constructed into a plant expression vector under the control of a promoter and the 3' UTR of the rice amylase 3D gene. The plant expression vector was introduced into rice embryogenic callus (Oryza sativa L. cv. Dongjin) via particle bombardment-mediated transformation. The integration and transcription of the ApxIIA439-801 gene were confirmed by using genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis of ApxIIA439-801 antigen protein in transgenic rice callus was confirmed by western blot analysis. The concentration of antigen protein in lyophilized samples of transgenic rice callus was 250 μg/g. Immunizing mice with protein extracts from transgenic plants intranasally elicited secretory IgA. These results demonstrate the feasibility of using a transgenic plant to elicit immune responses against A. pleuropneumoniae. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production.

    Science.gov (United States)

    Menassa, Rima; Zhu, Hong; Karatzas, Costas N; Lazaris, Anthoula; Richman, Alex; Brandle, Jim

    2004-09-01

    Spider dragline silk is a unique biomaterial and represents nature's strongest known fibre. As it is almost as strong as many commercial synthetic fibres, it is suitable for use in many industrial and medical applications. The prerequisite for such a widespread use is the cost-effective production in sufficient quantities for commercial fibre manufacturing. Agricultural biotechnology and the production of recombinant dragline silk proteins in transgenic plants offer the potential for low-cost, large-scale production. The purpose of this work was to examine the feasibility of producing the two protein components of dragline silk (MaSp1 and MaSp2) from Nephila clavipes in transgenic tobacco. Two different promoters, the enhanced CaMV 35S promoter (Kay et al., 1987) and a new tobacco cryptic constitutive promoter, tCUP (Foster et al., 1999) were used, in conjunction with a plant secretory signal (PR1b), a translational enhancer (alfalfa mosaic virus, AMV) and an endoplasmic reticulum (ER) retention signal (KDEL), to express the MaSp1 and MaSp2 genes in the leaves of transgenic plants. Both genes expressed successfully and recombinant protein accumulated in transgenic plants grown in both greenhouse and field trials.

  12. Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence.

    Science.gov (United States)

    Liu, Mao-Sen; Li, Hui-Chun; Chang, You-Min; Wu, Min-Tze; Chen, Long-Fang Oliver

    2011-09-01

    Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days' post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

  13. A fluorescent probe for imaging p53-MDM2 protein-protein interaction.

    Science.gov (United States)

    Liu, Zhenzhen; Miao, Zhenyuan; Li, Jin; Fang, Kun; Zhuang, Chunlin; Du, Lupei; Sheng, Chunquan; Li, Minyong

    2015-04-01

    In this article, we describe a no-wash small-molecule fluorescent probe for detecting and imaging p53-MDM2 protein-protein interaction based on an environment-sensitive fluorescent turn-on mechanism. After extensive biological examination, this probe L1 exhibited practical activity and selectivity in vitro and in cellulo.

  14. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    Science.gov (United States)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  15. Fluorescent IgG fusion proteins made in E. coli.

    Science.gov (United States)

    Luria, Yael; Raichlin, Dina; Benhar, Itai

    2012-01-01

    Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called "Inclonals." By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the "Inclonals" technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals.

  16. An Arabidopsis mitochondrial uncoupling protein confers tolerance to drought and salt stress in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Kevin Begcy

    Full Text Available BACKGROUND: Plants are challenged by a large number of environmental stresses that reduce productivity and even cause death. Both chloroplasts and mitochondria produce reactive oxygen species under normal conditions; however, stress causes an imbalance in these species that leads to deviations from normal cellular conditions and a variety of toxic effects. Mitochondria have uncoupling proteins (UCPs that uncouple electron transport from ATP synthesis. There is evidence that UCPs play a role in alleviating stress caused by reactive oxygen species overproduction. However, direct evidence that UCPs protect plants from abiotic stress is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Tolerances to salt and water deficit were analyzed in transgenic tobacco plants that overexpress a UCP (AtUCP1 from Arabidopsis thaliana. Seeds of AtUCP1 transgenic lines germinated faster, and adult plants showed better responses to drought and salt stress than wild-type (WT plants. These phenotypes correlated with increased water retention and higher gas exchange parameters in transgenic plants that overexpress AtUCP1. WT plants exhibited increased respiration under stress, while transgenic plants were only slightly affected. Furthermore, the transgenic plants showed reduced accumulation of hydrogen peroxide in stressed leaves compared with WT plants. CONCLUSIONS/SIGNIFICANCE: Higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. Our data support previous assumptions that UCPs reduce the imbalance of reactive oxygen species. Our data also suggest that UCPs may play a role in stomatal closure, which agrees with other evidence of a direct relationship between these proteins and photosynthesis. Manipulation of the UCP protein expression in mitochondria is a new avenue for crop improvement and may lead to crops with greater tolerance for challenging environmental conditions.

  17. Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins for use in risk assessments of transgenic crops.

    Science.gov (United States)

    Raybould, Alan; Kilby, Peter; Graser, Gerson

    2013-04-01

    Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence.

  18. On the Design of Low-Cost Fluorescent Protein Biosensors

    Science.gov (United States)

    Tolosa, Leah

    , magnetic beads, nanoparticles or quantum dots designed to form covalent bonds with amino groups, sulfhydryl groups, carboxylic groups and other reactive functionalities in amino acids. It is not uncommon to conduct combinations of techniques, for example, the introduction of fluorescent labels or probes to proteins require in many cases, site-directed mutagenesis followed by covalent bonding of the fluorescent dye. Accordingly, two or more proteins can be combined to create hybrid or fusion proteins with multiple or altered functions. Indeed, research involving the green fluorescent protein and fluorescent proteins of a variety of colors has expanded by leaps and bounds in the last decade. Because these fluorescent proteins can be genetically encoded in cells, it is possible to observe various cellular processes in vivo. However, this topic has been reviewed extensively in the literature and, thus, will not be expounded on in this chapter.

  19. The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins.

    Science.gov (United States)

    Bosze, Zs; Hiripi, L; Carnwath, J W; Niemann, H

    2003-10-01

    Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.

  20. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    Science.gov (United States)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  1. Folding and unfolding of a non-fluorescent mutant of green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Wielgus-Kutrowska, Beata [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Narczyk, Marta [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Buszko, Anna [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Bzowska, Agnieszka [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Clark, Patricia L [Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556-5670 (United States)

    2007-07-18

    Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and it remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to efficiently form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of guanidinium hydrochloride (GdnHCl). The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of 1,1'-bis(4-anilino-5-naphthalenesulphonic acid) (bis-ANS). Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is the formation of a molten globule, prone to aggregation.

  2. A Novel Impedimetric Microfluidic Analysis System for Transgenic Protein Cry1Ab Detection

    Science.gov (United States)

    Jin, Shunru; Ye, Zunzhong; Wang, Yixian; Ying, Yibin

    2017-01-01

    Impedimetric analysis method is an important tool for food safety detection. In this work, a novel impedimetric microfluidic analysis system consisted of a printed gold electrode chip and a microfluidic flow cell was developed for sensitive and selective detection of transgenic protein Cry1Ab. Anti-Cry1Ab aptamer coated magnetic beads were used to recognize transgenic protein Cry1Ab and form Cry1Ab-aptamer modified magnetic beads. After separation, the obtained Cry1Ab-aptamer modified magnetic beads were dissolved in 0.01 M mannitol and followed by injection into the microfluidic flow cell for impedimetric measurement. At the frequency of 358.3 Hz, the impedance signal shows a good linearity with the concentrations of Cry1Ab protein at a range from 0 to 0.2 nM, and the detection limit is 0.015 nM. The results demonstrate that the impedimetric microfluidic analysis system provides an alternative way to enable sensitive, rapid and specific detection of transgenic protein Cry1Ab. PMID:28251986

  3. Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure.

    Directory of Open Access Journals (Sweden)

    Tomonobu M Watanabe

    Full Text Available Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.

  4. Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response.

    Science.gov (United States)

    Wakasa, Yuhya; Takaiwa, Fumio

    2016-01-01

    Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail.

  5. Amyloid-like protein inclusions in tobacco transgenic plants.

    Directory of Open Access Journals (Sweden)

    Anna Villar-Piqué

    Full Text Available The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered β-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.

  6. Effect of gamma irradiation on nutritional components and Cry1Ab protein in the transgenic rice with a synthetic cry1Ab gene from Bacillus thuringiensis[Transgenic rice; Gamma irradiation; Nutritional components; Cry1Ab protein

    Energy Technology Data Exchange (ETDEWEB)

    Wu Dianxing E-mail: dxwu@zju.edu.cn; Ye Qingfu; Wang Zhonghua; Xia Yingwu

    2004-01-01

    The effects of gamma irradiation on the transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis were investigated. There was almost no difference in the content of the major nutritional components, i.e. crude protein, crude lipid, eight essential amino acids and total ash between the irradiated grains and the non-irradiated transgenic rice. However, the amounts of Cry1Ab protein and apparent amylose in the irradiated transgenic rice were reduced significantly by the doses higher than 200 Gy. In vivo observation showed that Cry1Ab protein contents also decreased in the fresh leaf tissues of survival seedlings after irradiation with 200 Gy or higher doses and showed inhibition of seedling growth. The results indicate that gamma irradiation might improve the quality of transgenic rice due to removal of the toxic Cry1Ab protein.

  7. Fluorescence microscopy of single autofluorescent proteins for cellular biology

    CERN Document Server

    Cognet, Laurent; Choquet, Daniel; Lounis, Brahim

    2002-01-01

    In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells.

  8. A Novel Reporter Rat Strain That Conditionally Expresses the Bright Red Fluorescent Protein tdTomato.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Igarashi

    Full Text Available Despite the strength of the Cre/loxP recombination system in animal models, its application in rats trails that in mice because of the lack of relevant reporter strains. Here, we generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualizes neurons including their projection fibers and spines without any histological enhancement. In addition, a transgenic rat line (FLAME that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic Cre mRNA into fertilized ova. Our rat reporter system will facilitate connectome studies as well as the visualization of the fine structures of genetically identified cells for long periods both in vivo and ex vivo. Furthermore, FLAME is an ideal model for organ transplantation research owing to improved traceability of cells/tissues.

  9. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. Reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. Reinhardtii.

  10. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    OpenAIRE

    Natalia V. Permyakova; Zagorskaya, Alla A.; Belavin, Pavel A.; Elena A. Uvarova; Nosareva, Olesya V.; Nesterov, Andrey E.; Novikovskaya, Anna A.; Evgeniy L. Zav’yalov; Mikhail P Moshkin; Deineko, Elena V.

    2015-01-01

    Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This co...

  11. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

    Felske, A; Vandieken, V; Pauling, B V

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is pro...

  12. Enhanced disease resistance and drought tolerance in transgenic rice plants overexpressing protein elicitors from Magnaporthe oryzae.

    Science.gov (United States)

    Wang, Zhenzhen; Han, Qiang; Zi, Qian; Lv, Shun; Qiu, Dewen; Zeng, Hongmei

    2017-01-01

    Exogenous application of the protein elicitors MoHrip1 and MoHrip2, which were isolated from the pathogenic fungus Magnaporthe oryzae (M. oryzae), was previously shown to induce a hypersensitive response in tobacco and to enhance resistance to rice blast. In this work, we successfully transformed rice with the mohrip1 and mohrip2 genes separately. The MoHrip1 and MoHrip2 transgenic rice plants displayed higher resistance to rice blast and stronger tolerance to drought stress than wild-type (WT) rice and the vector-control pCXUN rice. The expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes was also increased, suggesting that these two elicitors may trigger SA signaling to protect the rice from damage during pathogen infection and regulate the ABA content to increase drought tolerance in transgenic rice. Trypan blue staining indicated that expressing MoHrip1 and MoHrip2 in rice plants inhibited hyphal growth of the rice blast fungus. Relative water content (RWC), water usage efficiency (WUE) and water loss rate (WLR) were measured to confirm the high capacity for water retention in transgenic rice. The MoHrip1 and MoHrip2 transgenic rice also exhibited enhanced agronomic traits such as increased plant height and tiller number.

  13. Expression of Human Papillomavirus Type 16 L1 Protein in Transgenic Tobacco Plants

    Institute of Scientific and Technical Information of China (English)

    Hong-Li LIU; Wen-Sheng LI; Ting LEI; Jing ZHENG; Zheng ZHANG; Xiao-Fei YAN; Zhe-Zhi WANG; Yi-Li WANG; Lü-Sheng SI

    2005-01-01

    To develop a plant expression system for the production of the human papillomavirus type 16(HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55 nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV 16L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.

  14. Detection of transgenic and endogenous plant DNA fragments and proteins in the digesta, blood, tissues, and eggs of laying hens fed with phytase transgenic corn.

    Directory of Open Access Journals (Sweden)

    Qiugang Ma

    Full Text Available The trials were conducted to assess the effects of long-term feeding with phytase transgenic corn (PTC to hens on laying performance and egg quality, and investigate the fate of transgenic DNA and protein in digesta, blood, tissues, and eggs. Fifty-week old laying hens (n = 144 were fed with a diet containing 62.4% PTC or non-transgenic isogenic control corn (CC for 16 weeks. We observed that feeding PTC to laying hens had no adverse effect on laying performance or egg quality (P>0.05 except on yolk color (P<0.05. Transgenic phyA2 gene and protein were rapidly degraded in the digestive tract and were not detected in blood, heart, liver, spleen, kidney, breast muscle, and eggs of laying hens fed with diet containing PTC. It was concluded that performance of hens fed diets containing PTC, as measured by egg production and egg quality, was similar to that of hens fed diets formulated with CC. There was no evidence of phyA2 gene or protein translocation to the blood, tissues, and eggs of laying hens.

  15. Detection of protein-protein interactions in plants using bimolecular fluorescence complementation.

    Science.gov (United States)

    Bracha-Drori, Keren; Shichrur, Keren; Katz, Aviva; Oliva, Moran; Angelovici, Ruthie; Yalovsky, Shaul; Ohad, Nir

    2004-11-01

    Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.

  16. [Advances in effects of insecticidal crystal proteins released from transgenic Bt crops on soil ecology].

    Science.gov (United States)

    Zhou, Xue-Yong; Liu, Ning; Zhao, Man; Li, He; Zhou, Lang; Tang, Zong-Wen; Cao, Fei; Li, Wei

    2011-05-01

    With the large scale cultivation of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystal proteins in the world, the problem of environmental safety caused by these Bt crops has received extensive attention. These insecticidal crystal proteins can be released into the soil continuously in the growing period of Bt plants. If their accumulation of the insecticidal crystal proteins exceeds consumption by insect larvae and degradation by the environmental factors, these insecticidal crystal proteins could constitute a hazard to non-target insects and soil microbiota. There are three main ways to release insecticidal crystal proteins into soil for Bt plants: root exudates, pollen falling, and crop reside returning. The Bt insecticidal crystal proteins released into soil can be adsorbed rapidly by active soil particles and the absorption equilibrium attained within 1-3 h. The adsorption protects Bt insecticidal crystal proteins against soil microbial degradation or enzyme degradation, which leads to remarkable prolong of the persistence of insecticidal activity. The change of soil microorganism species is an important index for evaluating the effect of Bt plants on soil ecology. The research showed that these insecticidal crystal proteins released by the Bt plant root exudates or Bt organism had no toxicity to the soil earthworms, nematodes, protozoa, bacteria and fungi; however, it could reduce the mycelium length of the arbuscular mycorrhizal fungi (AMF) and restrain AMF to form invasion unit. The influencing degree of Bt protein on soil enzyme activity varied with the releasing modes or growth period of Bt crops. Bt Cry1Ab protein can be taken up from soil by parts of following crops; however, different results were obtained with different commercial kits. To better understand the soil ecological evaluation about the insecticidal crystal proteins released from transgenic Bt crops, this review provides a comprehensive overview about the release

  17. Risk Assessment of Synergism and Recombination on the Transgenic Plants Containing Viral Movement Protein and Replicase Genes

    Institute of Scientific and Technical Information of China (English)

    NIU Yan-bing; LI Gui-xin; WEN Rui; ZHOU Xue-ping

    2003-01-01

    The transgenic tobacco plants transformed with movement protein gene of Tomato mosaic virus (ToMV) or Tobacco mosaic virus (TMV) and partial replicase gene of Cucumber mosaic virus (CMV) P1 isolate (CMV-P1), were inoculated with Potato virus X, Potato virus Y, TMV and CMV isolate RB (CMVRB), respectively. Symptom observation showed there were no symptom differences in transgenic tobacco plants as compared with those in non-transgenic tobacco plants. ELISA also illustrated that the virus concentrations in the transgenic plants were similar to those in non-transgenic plants, indicating that no synergism is found in these plants. The transgenic tobacco plants expressing movement protein gene of ToMV or partial replicase gene of CMV-P1 were inoculated with TMV and CMV-RB, respectively. The local or systemic infected leaves were then used for elucidation of the possible virus recombination in transgenic plants with biological infectivity test, ELISA and immuno-capture RT-PCR. Within 16 months, no recombination was found between transformed genes and inoculated virus genomes. The research provides fundamental data for understanding of the possible risk of the transgenic plants expressing viral sequences.

  18. Murine leukemia virus (MLV replication monitored with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Bittner Alexandra

    2004-12-01

    Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

  19. Colorful protein-based fluorescent probes for collagen imaging.

    Directory of Open Access Journals (Sweden)

    Stijn J A Aper

    Full Text Available Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488. The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

  20. Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

    Science.gov (United States)

    Maruyama, Nobuyuki; Fujiwara, Keigo; Yokoyama, Kazunori; Cabanos, Cerrone; Hasegawa, Hisakazu; Takagi, Kyoko; Nishizawa, Keito; Uki, Yuriko; Kawarabayashi, Takeshi; Shouji, Mikio; Ishimoto, Masao; Terakawa, Teruhiko

    2014-10-01

    There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

  1. In vivo labelling of hippocampal beta-amyloid in triple-transgenic mice with a fluorescent acetylcholinesterase inhibitor released from nanoparticles.

    Science.gov (United States)

    Härtig, Wolfgang; Kacza, Johannes; Paulke, Bernd-Reiner; Grosche, Jens; Bauer, Ute; Hoffmann, Anke; Elsinghorst, Paul W; Gütschow, Michael

    2010-01-01

    The drastic loss of cholinergic projection neurons in the basal forebrain is a hallmark of Alzheimer's disease (AD), and drugs most frequently applied for the treatment of dementia include inhibitors of the acetylcholine-degrading enzyme acetylcholinesterase (AChE). This protein is known to act as a ligand of beta-amyloid (Abeta) in senile plaques, a further neuropathological sign of AD. Recently, we have shown that the fluorescent, heterodimeric AChE inhibitor PE154 allows for the histochemical staining of cortical Abeta plaques in triple-transgenic (TTG) mice with age-dependent beta-amyloidosis and tau hyperphosphorylation, an established animal model for aspects of AD. In the present study, we have primarily demonstrated the targeting of Abeta-immunopositive plaques with PE154 in vivo for 4 h up to 1 week after injection into the hippocampi of 13-20-month-old TTG mice. Numerous plaques, double-stained for PE154 and Abeta-immunoreactivity, were revealed by confocal laser-scanning microscopy. Additionally, PE154 targeted hippocampal Abeta deposits in aged TTG mice after injection of carboxylated polyglycidylmethacrylate nanoparticles delivering the fluorescent marker in vivo. Furthermore, biodegradable core-shell polystyrene/polybutylcyanoacrylate nanoparticles were found to be suitable, alternative vehicles for PE154 as a useful in vivo label of Abeta. Moreover, we were able to demonstrate that PE154 targeted Abeta, but neither phospho-tau nor reactive astrocytes surrounding the plaques. In conclusion, nanoparticles appear as versatile carriers of AChE inhibitors and other promising drugs for the treatment of AD.

  2. Protein immobilization and fluorescence quenching on polydopamine thin films.

    Science.gov (United States)

    Chen, Daqun; Zhao, Lei; Hu, Weihua

    2016-09-01

    Mussel inspired polydopamine (PDA) film has attracted great interest as a versatile functional coating for biomolecule immobilization in various bio-related devices. However, the details regarding the interaction between a protein and PDA film remain unclear. Particularly, there is very limited knowledge regarding the protein immobilization on PDA film, even though it is of essential importance in various fields. The situation is even more complicated if considering the fact that quite a number of approaches (e.g., different oxidizing reagent, buffer pH, grown time, grown media, etc.) have been developed to grow PDA films. In this work, protein attachment on PDA film was systematically investigated by using the real-time and label-free surface plasmon resonance (SPR) technique. The kinetics of protein-PDA interaction was explored and the influence of buffer pH and deposition media on the protein attachment was studied. Fluorescent protein microarray was further printed on PDA-coated glass slides for quantitative investigations and together with SPR data, the interesting fluorescence quenching phenomenon of PDA film was revealed. This work may deepen our understanding on the PDA-protein interaction and offer a valuable guide for efficient protein attachment on PDA film in various bio-related applications.

  3. Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

    Science.gov (United States)

    Moseyko, N.; Feldman, L. J.

    2001-01-01

    This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non-invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH-sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH-sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole-root tissues of A. thaliana is reported. The utility of pH-sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.

  4. POLA EKSPRESI GEN ENHANCED GREEN FLUORESCENT PROTEIN PADA EMBRIO DAN LARVA IKAN PATIN SIAM (Pangasianodon hypophthalmus

    Directory of Open Access Journals (Sweden)

    Raden Roro Sri Pudji Sinarni Dewi

    2016-04-01

    menggunakan gen reporter berguna untuk mendesain konstruksi gen yang akan digunakan pada penelitian transgenesis. Gen reporter yang umum digunakan dalam penelitian ekspresi sementara transgen adalah gen GFP (green fluorescent protein. Pengamatan gen EGFP (enhanced green fluorescent protein pada embrio dan larva ikan patin siam (Pangasianodon hypophthalmus ditujukan untuk mendapatkan informasi mengenai kemampuan promoter -aktin ikan mas dalam mengendalikan ekspresi gen EGFP. Gen EGFP diintroduksikan ke dalam sperma ikan patin siam menggunakan metode elektroporasi. Sperma yang telah dielektroporasi digunakan untuk membuahi sel telur ikan patin siam. Pengamatan ekspresi gen EGFP dilakukan setiap enam jam dimulai dari embrio fase 2 sel sampai larva. Berdasarkan hasil penelitian, gen EGFP terekspresi pada fase embrio dan larva ikan patin siam. Puncak ekspresi gen EGFP terjadi pada fase neurula dan menurun pada fase larva. Berdasarkan penelitian ini maka ikan patin siam transgenik telah berhasil dibentuk dan promoter -aktin ikan mas terbukti aktif dalam mengarahkan ekspresi gen asing (GFP di dalam tubuh ikan patin siam.

  5. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

    2008-04-15

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91(phox). By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91(phox) are approximately 1.38 and approximately 1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.

  6. Colloidal quantum dots for fluorescent labels of proteins

    Science.gov (United States)

    Gladyshev, P.; Kouznetsov, V.; Martinez Bonilla, C.; Dezhurov, S.; Krilsky, D.; Vasiliev, A.; Morenkov, O.; Vrublevskaya, V.; Tsygankov, P.; Ibragimova, S.; Rybakova, A.

    2016-10-01

    The work is devoted to the synthesis of colloidal quantum dots (QDs) and their bioconjugates with proteins. Various QDs were obtained as well with synthesis method in an organic solvent followed by hydrophilization and functionalization or synthesis in aqueous phase provides obtaining hydrophilic QDs directly. Particular attention is paid to the synthesis of QDs as fluorescent tags in the near infrared where minimum absorption occurs and the fluorescence of biological tissue and synthetic materials used in analytical systems. A method for the QDs synthesis of type fluorescent core/shell CdTeSe/CdS/CdZnS-PolyT with mixed telluride, selenide cadmium core with a high quantum yield and high resistance to photoaging. It is shown that these quantum dots may be effectively used in the immunoassay.

  7. Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats

    Directory of Open Access Journals (Sweden)

    Guidot David M

    2008-04-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 infection and the consequent acquired immunodeficiency syndrome (AIDS has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef. Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1 and Transforming Growth Factor-β1 (TGFβ1, three factors associated with muscle catabolism. Results Consistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss, the oxidized form of the anti-oxidant cysteine (Cys, and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold and TGFβ1 (~5-fold and ~3-fold in heart and

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

    Science.gov (United States)

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

    2017-01-01

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

  9. A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

    Science.gov (United States)

    Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase…

  10. Quantitative Analysis of Spatial Protein-protein Proximity in Fluorescence Confocal Microscopy

    Science.gov (United States)

    Wu, Yong; Liu, Yi-Kuang; Eghbali, Mansoureh; Stefani, Enrico

    2009-02-01

    To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component.

  11. Fluorescent biosensors for high throughput screening of protein kinase inhibitors.

    Science.gov (United States)

    Prével, Camille; Pellerano, Morgan; Van, Thi Nhu Ngoc; Morris, May C

    2014-02-01

    High throughput screening assays aim to identify small molecules that interfere with protein function, activity, or conformation, which can serve as effective tools for chemical biology studies of targets involved in physiological processes or pathways of interest or disease models, as well as templates for development of therapeutics in medicinal chemistry. Fluorescent biosensors constitute attractive and powerful tools for drug discovery programs, from high throughput screening assays, to postscreen characterization of hits, optimization of lead compounds, and preclinical evaluation of candidate drugs. They provide a means of screening for inhibitors that selectively target enzymatic activity, conformation, and/or function in vitro. Moreover, fluorescent biosensors constitute useful tools for cell- and image-based, multiplex and multiparametric, high-content screening. Application of fluorescence-based sensors to screen large and complex libraries of compounds in vitro, in cell-based formats or whole organisms requires several levels of optimization to establish robust and reproducible assays. In this review, we describe the different fluorescent biosensor technologies which have been applied to high throughput screens, and discuss the prerequisite criteria underlying their successful application. Special emphasis is placed on protein kinase biosensors, since these enzymes constitute one of the most important classes of therapeutic targets in drug discovery.

  12. The trafficking pathway of a wheat storage protein in transgenic rice endosperm

    Science.gov (United States)

    Oszvald, Maria; Tamas, Laszlo; Shewry, Peter R.; Tosi, Paola

    2014-01-01

    Background and Aims The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these. Methods The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies. Key results The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells. Conclusions The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm. PMID:24603605

  13. Green fluorescent protein is lighting up fungal biology

    Science.gov (United States)

    Lorang, J.M.; Tuori, R.P; Martinez, J.P; Sawyer, T.L.; Redman, R.S.; Rollins, J. A.; Wolpert, T.J.; Johnson, K.B.; Rodriguez, R.J.; Dickman, M. B.; Ciuffetti, L.M.

    2001-01-01

    Prasher (42) cloned a cDNA for the green fluorescent protein (GFP) gene from the jellyfishAequorea victoria in 1992. Shortly thereafter, to the amazement of many investigators, this gene or derivatives thereof were successfully expressed and conferred fluorescence to bacteria andCaenorhabditis elegans cells in culture (10,31), followed by yeast (24, 39), mammals (40), Drosophila (66),Dictyostelium(23, 30), plants (28,49), and filamentous fungi (54). The tremendous success of GFP as a reporter can be attributed to unique qualities of this 238-amino-acid, 27-kDa protein which absorbs light at maxima of 395 and 475 nm and emits light at a maximum of 508 nm. The fluorescence of GFP requires only UV or blue light and oxygen, and therefore, unlike the case with other reporters (β-glucuronidase, β-galacturonidase, chloramphenicol acetyltransferase, and firefly luciferase) that rely on cofactors or substrates for activity, in vivo observation ofgfp expression is possible with individual cells, with cell populations, or in whole organisms interacting with symbionts or environments in real time. Complications caused by destructive sampling, cell permeablization for substrates, or leakage of products do not occur. Furthermore, the GFP protein is extremely stable in vivo and has been fused to the C or N terminus of many cellular and extracellular proteins without a loss of activity, thereby permitting the tagging of proteins for gene regulation analysis, protein localization, or specific organelle labeling. The mature protein resists many proteases and is stable up to 65°C and at pH 5 to 11, in 1% sodium dodecyl sulfate or 6 M guanidinium chloride (reviewed in references 17and 67), and in tissue fixed with formaldehyde, methanol, or glutaraldehyde. However, GFP loses fluorescence in methanol-acetic acid (3:1) and can be masked by autofluorescent aldehyde groups in tissue fixed with glutaraldehyde. Fluorescence is optimal at pH 7.2 to 8.0 (67).

  14. The fluorescent protein palette: tools for cellular imaging†

    Science.gov (United States)

    Davidson, Michael W.

    2010-01-01

    This critical review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biology and physiology. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quantitative measurements in living systems. “If wood is rubbed with the Pulmo marinus, it will have all the appearance of being on fire; so much so, indeed, that a walking-stick, thus treated, will light the way like a torch” (translation of Pliny the Elder from John Bostock, 1855). PMID:19771335

  15. Improved "optical highlighter" probes derived from discosoma red fluorescent protein.

    Science.gov (United States)

    Robinson, Lisbeth C; Marchant, Jonathan S

    2005-02-01

    The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.

  16. Protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves.

    Science.gov (United States)

    Schweiger, Regina; Schwenkert, Serena

    2014-03-09

    Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

  17. Nucleic acid and protein elimination during the sugar manufacturing process of conventional and transgenic sugar beets.

    Science.gov (United States)

    Klein, J; Altenbuchner, J; Mattes, R

    1998-02-26

    The fate of cellular DNA during the standard purification steps of the sugar manufacturing process from conventional and transgenic sugar beets was determined. Indigenous nucleases of sugar beet cells were found to be active during the first extraction step (raw juice production) which was carried out at 70 degrees C. This and the consecutive steps of the manufacturing process were validated in terms of DNA degradation by competitive PCR of added external DNA. Each step of the process proved to be very efficient in the removal of nucleic acids. Taken together, the purification steps have the potential to reduce the amount of DNA by a factor of > 10(14), exceeding by far the total amount of DNA present in sugar beets. Furthermore, the gene products of the transgenes neomycin phosphotransferase and BNYVV (rhizomania virus) coat protein CP21 were shown to be removed during the purification steps, so that they could not be detected in the resulting white sugar. Thus, sugar obtained from conventional and transgenic beets is indistinguishable or substantially equivalent with respect to purity.

  18. Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters

    Science.gov (United States)

    2016-01-13

    and β-sheet structure present in the stabilizing protein. These data support the hypothesis that the pressure -induced fluorescence enhancement...ARL-TR-7572 ● JAN 2016 US Army Research Laboratory Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold...JAN 2016 US Army Research Laboratory Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters by Abby

  19. Monomeric red fluorescent protein variants used for imaging studies in different species

    NARCIS (Netherlands)

    Mueller-Taubenberger, Annette; Vos, Michel J.; Boettger, Angelika; Lasi, Margherita; Lai, Frank P. L.; Fischer, Markus; Rottner, Klemens

    2006-01-01

    Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation

  20. An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    Jun Zhou; Jian Lin; Cuihong Zhou; Xiaoyan Deng; Bin Xia

    2011-01-01

    Bimolecular fluorescence complementation (BiFC) has been widely used in the analysis of protein-protein interactions (PPIs) in recent years. There are many notable advantages of BiFC such as convenience and direct visualization of PPI in cells. However, BiFC has one common limitation: the separated non-fluorescent fragments can be spontaneously self-assembled into an intact protein,which leads to false-positive results. In this study, a pair of complementary fragments (sfGFPN and sfGFPC) was constructed by splitting superfolder GFP (sfGFP) between the 214 and 215 amino acid residue, and sfGFPC was mutated by site-directed gene mutagenesis to decrease the signal of negative control. Our results showed that mutations in sfGFPC (sfGFPC(m12)) can effectively decrease the signal of negative control. Thus, we provide an improved BiFC tool for the analysis of PPI. Further,since the self-assembly problem is a common shortcoming for application of BiFC, our research provides a feasible strategy for other BiFC candidate proteins with the same problem.

  1. Detection of transgenic and endogenous plant DNA fragments and proteins in the digesta, blood, tissues, and eggs of laying hens fed with phytase transgenic corn.

    Science.gov (United States)

    Ma, Qiugang; Gao, Chunqi; Zhang, Jianyun; Zhao, Lihong; Hao, Wenbo; Ji, Cheng

    2013-01-01

    The trials were conducted to assess the effects of long-term feeding with phytase transgenic corn (PTC) to hens on laying performance and egg quality, and investigate the fate of transgenic DNA and protein in digesta, blood, tissues, and eggs. Fifty-week old laying hens (n = 144) were fed with a diet containing 62.4% PTC or non-transgenic isogenic control corn (CC) for 16 weeks. We observed that feeding PTC to laying hens had no adverse effect on laying performance or egg quality (P>0.05) except on yolk color (PTransgenic phyA2 gene and protein were rapidly degraded in the digestive tract and were not detected in blood, heart, liver, spleen, kidney, breast muscle, and eggs of laying hens fed with diet containing PTC. It was concluded that performance of hens fed diets containing PTC, as measured by egg production and egg quality, was similar to that of hens fed diets formulated with CC. There was no evidence of phyA2 gene or protein translocation to the blood, tissues, and eggs of laying hens.

  2. LeCDJ1, a chloroplast DnaJ protein, facilitates heat tolerance in transgenic tomatoes

    Institute of Scientific and Technical Information of China (English)

    Fanying Kong; Yongsheng Deng; Guodong Wang; Jieru Wang; Xiaoqing Liang; Qingwei Meng

    2014-01-01

    The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1) were investigat-ed using wild-type (WT) and sense transgenic tomatoes. The LeCDJ1 expression was upregulated by 38 °C, 42 °C, 45 °C, NaCl, PEG, methyl viologen (MV) and hydrogen peroxide (H2O2), but not by 30 °C and 35 °C. Meanwhile, LeCDJ1 was involved in the response of plants to abscisic acid (ABA). Under heat stress, the sense plants showed better growth, higher chlorophyll content, lower malondialdehyde (MDA) accumulation and relative electrical conductivity (REC), and also less PSII photoinhibition than WT. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher maximum photo-chemistry efficiency of PSII (Fv/Fm) and D1 protein levels than the SM-untreated WT, suggesting that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, the relatively lower super-oxide radical (O2•?) and H2O2 levels in the sense plants were considered to be due to the higher ascorbate peroxidase (APX) and superoxide dismutase (SOD) activity, which seemed unlikely dependent on their transcription level. These results indicated that LeCDJ1 overexpression facilitated heat tolerance in transgenic tomatoes.

  3. Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis.

    Science.gov (United States)

    Zhang, Li; Salom, David; He, Jianhua; Okun, Alex; Ballesteros, Juan; Palczewski, Krzysztof; Li, Ning

    2005-11-08

    G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signaling proteins; however, the only known GPCR crystal structure is that of rhodopsin. This disparity reflects the difficulty in generating purified GPCR samples of sufficient quantity and quality. Rhodopsin, the light receptor of retinal rod neurons, is produced in large amounts of homogeneous quality in the vertebrate retina. We used transgenic Xenopus laevis to convert these retina rod cells into bioreactors to successfully produce 20 model GPCRs. The receptors accumulated in rod outer segments and were homogeneously glycosylated. Ligand and [(35)S]GTPgammaS binding assays of the 5HT(1A) and EDG(1) GPCRs confirmed that they were properly folded and functional. 5HT(1A)R was highly purified by taking advantage of the rhodopsin C-terminal immunoaffinity tag common to all GPCR constructs. We have also developed an automated system that can generate hundreds of transgenic tadpoles per day. This expression approach could be extended to other animal model systems and become a general method for the production of large numbers of GPCRs and other membrane proteins for pharmacological and structural studies.

  4. Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.

    Science.gov (United States)

    Chen, Shengxi; Fahmi, Nour Eddine; Bhattacharya, Chandrabali; Wang, Lin; Jin, Yuguang; Benkovic, Stephen J; Hecht, Sidney M

    2013-11-26

    In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational

  5. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  6. In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

    Science.gov (United States)

    Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

    2014-03-01

    Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

  7. Simultaneous time-lamination imaging of protein association using a split fluorescent timer protein.

    Science.gov (United States)

    Takamura, Ayari; Hattori, Mitsuru; Yoshimura, Hideaki; Ozawa, Takeaki

    2015-03-17

    Studies of temporal behaviors of protein association in living cells are crucially important for elucidating the fundamental roles and the mechanism of interactive coordination for cell activities. We developed a method for investigating the temporal alternation of a particular protein assembly using monomeric fluorescent proteins, fluorescent timers (FTs), of which the fluorescent color changes from blue to red over time. We identified a dissection site of the FTs, which allows complementation of the split FT fragments. The split fragments of each FT variant recovered their fluorescence and maintained inherent rates of the color changes upon the reassembly of the fragments in vitro. We applied this method to visualize the aggregation process of α-synuclein in living cells. The size of the aggregates with the temporal information was analyzed from ratio values of the blue and red fluorescence of the reconstituted FTs, from which the aggregation rates were evaluated. This method using the split FT fragments enables tracing and visualizing temporal alternations of various protein associations by single fluorescence measurements at a given time point.

  8. Total Soluble Protein Extraction for Improved Proteomic Analysis of Transgenic Rice Plant Roots.

    Science.gov (United States)

    Raorane, Manish L; Narciso, Joan O; Kohli, Ajay

    2016-01-01

    With the advent of high-throughput platforms, proteomics has become a powerful tool to search for plant gene products of agronomic relevance. Protein extractions using multistep protocols have been shown to be effective to achieve better proteome profiles than simple, single-step extractions. These protocols are generally efficient for above ground tissues such as leaves. However, each step leads to loss of some amount of proteins. Additionally, compounds such as proteases in the plant tissues lead to protein degradation. While protease inhibitor cocktails are available, these alone do not seem to suffice when roots are included in the plant sample. This is obvious given the lack of high molecular weight (HMW) proteins obtained from samples that include root tissue. For protein/proteome analysis of transgenic plant roots or of seedlings, which include root tissue, such pronounced protein degradation is especially undesirable. A facile protein extraction protocol is presented, which ensures that despite the inclusion of root tissues there is minimal loss in total protein components.

  9. The labeling of C57BL/6j derived embryonic stem cells with enhanced green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    滕路; 张崇本; 尤洁芳; 尚克刚; 顾军

    2003-01-01

    Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the "green" ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

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

    Science.gov (United States)

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

    2008-01-01

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

  11. Intracellular distribution of cowpea mosaic virus movement protein as visualised by green fluorescent protein fusions

    NARCIS (Netherlands)

    Gopinath, K.; Bertens, P.; Pouwels, J.; Marks, H.; Lent, van J.W.M.; Wellink, J.E.; Kammen, van A.

    2003-01-01

    Cowpea mosaic virus (CPMV) derivatives expressing movement protein (MP) green fluorescent protein (GFP) fusions (MP:GFP) were used to study the intracellular targeting and localization of the MP in cowpea protoplasts and plants. In protoplasts, a virus coding for a wild type MP:GFP (MPfGFP) induced

  12. Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G-Quadruplexes.

    Science.gov (United States)

    Dang, Dung Thanh; Phan, Anh Tuân

    2016-01-01

    We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.

  13. Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants.

    Science.gov (United States)

    Palencia, Edwin Rene; Glenn, Anthony Elbie; Hinton, Dorothy Mae; Bacon, Charles Wilson

    2013-09-01

    Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli-maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP1) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri. © 2013.

  14. [Transgenic tobacco plants with ribosome inactivating protein gene cassin from Cassia occidentalis and their resistance to tobacco mosaic virus].

    Science.gov (United States)

    Ruan, Xiao-Lei; Liu, Li-Fang; Li, Hua-Ping

    2007-12-01

    Cassin, the new gene of ribosome-inactivating protein (RIP) isolated from Cassia occidentalis, was inserted into expression vector pBI121 to produce plant expression vector pBI121-cassin (Figs.1, 2). pBI121-cassin was introduced into tobacco cultivar 'K326' by the Agrobacteriurm tumefaciens transformation method and more than 100 independent transformants were obtained. Southern blot hybridization analysis showed that a single gene locus was inserted into the chromosome of the transgenic tobacco lines (Fig.5) and PCR analysis of segregation population of progeny indicated that the inheritance of transgene was dominant in transgenic lines (Fig.4, Table 1). Results of RT-PCR and Northern blot hybridization analysis showed that transgene could be transcribed correctly (Figs.5, 6) . Three self-pollination lines of transgenic T(1) and T(2) were challenged with TMV at different concentration titers by mechanical inoculation. The transgenic lines exhibited different levels of resistance to TMV with the nontransgenic plants. After both titers of TMV concentration were inoculated, transgenic lines were considered as the highly resistant type with a delay of 4-13 d in development of symptoms and 10%-25% of test plants were infected, while nontransgenic control plants were susceptible typical symptoms on the newly emerged leaves (Table 2). One T(2) line, T(2)-8-2-1, was regarded as an immune type because it did not show any symptoms during 70 d and all plants were shown to be virus free by ELISA tests.

  15. Intrinsic fluorescence of protein in turbid media using empirical relation based on Monte Carlo lookup table

    Science.gov (United States)

    Einstein, Gnanatheepam; Udayakumar, Kanniyappan; Aruna, Prakasarao; Ganesan, Singaravelu

    2017-03-01

    Fluorescence of Protein has been widely used in diagnostic oncology for characterizing cellular metabolism. However, the intensity of fluorescence emission is affected due to the absorbers and scatterers in tissue, which may lead to error in estimating exact protein content in tissue. Extraction of intrinsic fluorescence from measured fluorescence has been achieved by different methods. Among them, Monte Carlo based method yields the highest accuracy for extracting intrinsic fluorescence. In this work, we have attempted to generate a lookup table for Monte Carlo simulation of fluorescence emission by protein. Furthermore, we fitted the generated lookup table using an empirical relation. The empirical relation between measured and intrinsic fluorescence is validated using tissue phantom experiments. The proposed relation can be used for estimating intrinsic fluorescence of protein for real-time diagnostic applications and thereby improving the clinical interpretation of fluorescence spectroscopic data.

  16. The mungbean yellow mosaic begomovirus transcriptional activator protein transactivates the viral promoter-driven transgene and causes toxicity in transgenic tobacco plants.

    Science.gov (United States)

    Rajeswaran, Rajendran; Sunitha, Sukumaran; Shivaprasad, Padubidri V; Pooggin, Mikhail M; Hohn, Thomas; Veluthambi, Karuppannan

    2007-12-01

    The Begomovirus transcriptional activator protein (TrAP/AC2/C2) is a multifunctional protein which activates the viral late gene promoters, suppresses gene silencing, and determines pathogenicity. To study TrAP-mediated transactivation of a stably integrated gene, we generated transgenic tobacco plants with a Mungbean yellow mosaic virus (MYMV) AV1 late gene promoter-driven reporter gene and supertransformed them with the MYMV TrAP gene driven by a strong 35S promoter. We obtained a single supertransformed plant with an intact 35S-TrAP gene that activated the reporter gene 2.5-fold. However, 10 of the 11 supertransformed plants did not have the TrAP region of the T-DNA, suggesting the likely toxicity of TrAP in plants. Upon transformation of wild-type tobacco plants with the TrAP gene, six of the seven transgenic plants obtained had truncated T-DNAs which lacked TrAP. One plant, which had the intact TrAP gene, did not express TrAP. The apparent toxic effect of the TrAP transgene was abolished by mutations in its nuclear-localization signal or zinc-finger domain and by deletion of its activation domain. Therefore, all three domains of TrAP, which are required for transactivation and suppression of gene silencing, also are needed for its toxic effect.

  17. Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

    NARCIS (Netherlands)

    Meade-White, K.; Race, B.; Trifilo, M.; Bossers, A.; Favara, C.; Lacasse, R.; Miller, M.; Williams, E.; Oldstone, M.; Race, R.; Chesebro, B.

    2007-01-01

    Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer

  18. Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

    Science.gov (United States)

    Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed in detail by quantitative 2...

  19. Deposition of BACE-1 Protein in the Brains of APP/PS1 Double Transgenic Mice

    Directory of Open Access Journals (Sweden)

    Gang Luo

    2016-01-01

    Full Text Available The main causes of Alzheimer’s disease remain elusive. Previous data have implicated the BACE-1 protein as a central player in the pathogenesis of Alzheimer’s disease. However, many inhibitors of BACE-1 have failed during preclinical and clinical trials for AD treatment. Therefore, uncovering the exact role of BACE-1 in AD may have significant impact on the future development of therapeutic agents. Three- and six-month-old female APP/PS1 double transgenic mice were used to study abnormal accumulation of BACE-1 protein in brains of mice here. Immunofluorescence, immunohistochemistry, and western blot were performed to measure the distributing pattern and expression level of BACE-1. We found obvious BACE-1 protein accumulation in 3-month-old APP/PS1 mice, which had increased by the time of 6 months. Coimmunostaining results showed BACE-1 surrounded amyloid plaques in brain sections. The abnormal protein expression might not be attributable to the upregulation of BACE-1 protein, as no significant difference of protein expression was observed between wild-type and APP/PS1 mice. With antibodies against BACE-1 and CD31, we found a high immunoreactive density of BACE-1 protein on the outer layer of brain blood vessels. The aberrant distribution of BACE-1 in APP/PS1 mice suggests BACE-1 may be involved in the microvascular abnormality of AD.

  20. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  1. Strong seed-specific protein expression from the Vigna radiata storage protein 8SGα promoter in transgenic Arabidopsis seeds.

    Science.gov (United States)

    Chen, Mo-Xian; Zheng, Shu-Xiao; Yang, Yue-Ning; Xu, Chao; Liu, Jie-Sheng; Yang, Wei-Dong; Chye, Mee-Len; Li, Hong-Ye

    2014-03-20

    Vigna radiata (mung bean) is an important crop plant and is a major protein source in developing countries. Mung bean 8S globulins constitute nearly 90% of total seed storage protein and consist of three subunits designated as 8SGα, 8SGα' and 8SGβ. The 5'-flanking sequences of 8SGα' has been reported to confer high expression in transgenic Arabidopsis seeds. In this study, a 472-bp 5'-flanking sequence of 8SGα was identified by genome walking. Computational analysis subsequently revealed the presence of numerous putative seed-specific cis-elements within. The 8SGα promoter was then fused to the gene encoding β-glucuronidase (GUS) to create a reporter construct for Arabidopsis thaliana transformation. The spatial and temporal expression of 8SGα∷GUS, as investigated using GUS histochemical assays, showed GUS expression exclusively in transgenic Arabidopsis seeds. Quantitative GUS assays revealed that the 8SGα promoter showed 2- to 4-fold higher activity than the Cauliflower Mosaic Virus (CaMV) 35S promoter. This study has identified a seed-specific promoter of high promoter strength, which is potentially useful for directing foreign protein expression in seed bioreactors.

  2. Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

    Directory of Open Access Journals (Sweden)

    Huxley Clare

    2002-10-01

    Full Text Available Abstract Background Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM, a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1 gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases plays an essential role in the retinal degenerative process. Results To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal. Conclusions We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.

  3. Development of fiber optic spectroscopy for in-vitro and in-planta detection of fluorescent proteins

    Science.gov (United States)

    Liew, Oi Wah; Chen, Jun-Wei; Asundi, Anand K.

    2001-10-01

    The objective of this project is to apply photonics technology to bio-safety management of genetically modified (GM) plants. The conventional method for screening GM plants is through selection using antibiotic resistance markers. There is public concern with such approaches and these are associated with food safety issues, escape of antibiotic resistance genes to pathogenic microorganisms and interference with antibiotic therapy. Thus, the strategy taken in this project is to replace antibiotic resistance markers with fluorescent protein markers that allow for rapid and non-invasive optical screening of genetically modified plants. In this paper, fibre optic spectroscopy was developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in planta. In vitro detection was first carried out to optimize the sensitivity of the optical system. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fibre optic spectroscopy using different light sources, namely, blue LED (475 nm), tungsten halogen (350 - 1000 nm) and double frequency Nd:YAG green laser (532 nm). Fluorescence near the expected emission wavelengths could be detected up to 320X dilution for EGFP and DsRED with blue LED and 532 nm green laser, respectively, as the excitation source. Tungsten halogen was found to be unsuitable for excitation of both EGFP and DsRED. EGFP was successfully purified by size separation under non-denaturing electrophoretic conditions and quantified. The minimum concentration of EGFP detectable with blue LED excitation was 5 mg/ml. To determine the capability of spectroscopy detection in planta, transgenic potato hairy roots and whole modified plant lines expressing the

  4. Expression of plant sweet protein brazzein in the milk of transgenic mice.

    Directory of Open Access Journals (Sweden)

    Sen Yan

    Full Text Available Sugar, the most popular sweetener, is essential in daily food. However, excessive sugar intake has been associated with several lifestyle-related diseases. Finding healthier and more economical alternatives to sugars and artificial sweeteners has received increasing attention to fulfill the growing demand. Brazzein, which comes from the pulp of the edible fruit of the African plant Pentadiplandra brazzeana Baill, is a protein that is 2,000 times sweeter than sucrose by weight. Here we report the production of transgenic mice that carry the optimized brazzein gene driven by the goat Beta-casein promoter, which specifically directs gene expression in the mammary glands. Using western blot analysis and immunohistochemistry, we confirmed that brazzein could be efficiently expressed in mammalian milk, while retaining its sweetness. This study presents the possibility of producing plant protein-sweetened milk from large animals such as cattle and goats.

  5. Binding phenomena and fluorescence quenching. II: Photophysics of aromatic residues and dependence of fluorescence spectra on protein conformation

    Science.gov (United States)

    Callis, Patrik R.

    2014-12-01

    The three amino acids with aromatic ring side chains-phenylalanine (Phe), tyrosine (Tyr), and especially tryptophan (Trp) have played a long and productive role in helping unlock the secrets of protein behavior by optical spectroscopy (absorption, fluorescence, circular dichroism, etc.) In principle, an appropriately placed Trp will undergo fluorescence wavelength and/or intensity changes upon whatever functional process a protein performs. Although perceived to be enigmatic and not well understood, Trp is arguably now better understood than many of the extrinsic probes currently in use. Basic principles of intrinsic tryptophan fluorescence quenching and wavelength shifts in proteins are presented, with strong emphasis on the importance of electrostatics. The condensed description of findings from recent experiments and simulations of tryptophan fluorescence and intrinsic quenching in proteins is designed to help authors in planning and interpreting experimental results of ligand binding studies.

  6. Synaptotrophic effects of human amyloid beta protein precursors in the cortex of transgenic mice.

    Science.gov (United States)

    Mucke, L; Masliah, E; Johnson, W B; Ruppe, M D; Alford, M; Rockenstein, E M; Forss-Petter, S; Pietropaolo, M; Mallory, M; Abraham, C R

    1994-12-15

    The amyloid precursor protein (APP) is involved in Alzheimer's disease (AD) because its degradation products accumulate abnormally in AD brains and APP mutations are associated with early onset AD. However, its role in health and disease appears to be complex, with different APP derivatives showing either neurotoxic or neurotrophic effects in vitro. To elucidate the effects APP has on the brain in vivo, cDNAs encoding different forms of human APP (hAPP) were placed downstream of the neuron-specific enolase (NSE) promoter. In multiple lines of NSE-hAPP transgenic mice neuronal overexpression of hAPP was accompanied by an increase in the number of synaptophysin immunoreactive (SYN-IR) presynaptic terminals and in the expression of the growth-associated marker GAP-43. In lines expressing moderate levels of hAPP751 or hAPP695, this effect was more prominent in homozygous than in heterozygous transgenic mice. In contrast, a line with several-fold higher levels of hAPP695 expression showed less increase in SYN-IR presynaptic terminals per amount of hAPP expressed than the lower expressor lines and a decrease in synaptotrophic effects in homozygous compared with heterozygous offspring. Transgenic mice (2-24 months of age) showed no evidence for amyloid deposits or neurodegeneration. These findings suggest that APP may be important for the formation/maintenance of synapses in vivo and that its synaptotrophic effects may be critically dependent on the expression levels of different APP isoforms. Alterations in APP expression, processing or function could contribute to the synaptic pathology seen in AD.

  7. Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis, streptavidin-binding peptide (SBP, and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM. These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

  8. Quantitative Determination of Spatial Protein-protein Proximity in Fluorescence Confocal Microscopy

    CERN Document Server

    Wu, Yong; Ou, Jimmy; Li, Min; Toro, Ligia; Stefani, Enrico

    2009-01-01

    To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component. This new method is illustrated by analyzing colocalization in both simulated and biological images.

  9. Recombination with coat protein transgene in a complemen-tation system based on Cucumber mosaic virus (CMV)

    Institute of Scientific and Technical Information of China (English)

    LEI; Wanli

    2001-01-01

    [1]Palukaitis, P., Roossinck, M. J., Dietzgen, R. G. et al., Cucumber mosaic virus, Adv. Virus Res., 1992, 41: 281-348.[2]Hayes, R. J., Buck, K. W., Complete replication of an eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase, Cell, 1990, 63: 363-369.[3]Nitta, N., Takanami, Y., Kuwata, S. et al., Inoculation with RNAs 1 and 2 of cucumber mosaic virus induces viral RNA replicase activity in tobacco mesophyll protoplasts, J. Gen. Virol., 1988, 69: 2695-2700.[4]Suzuki, M., Kuwata, S., Kataoda, J. et al., Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system, Virology, 1991, 183: 106-113.[5]Canto, T., Prior, D. A. M., Hellwald, K. H. et al., Characterization of cucumber mosaic virus (IV)--Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus, Virology, 1997, 237:237-248.[6]Takanami, Y., A striking change in symptoms on cucumber mosaic virus-infected tobacco plants induced by a satellite RNA, Virology, 1981, 109: 120-126.[7]DeBorde, D. C., Naeve, C. W., Herlocher, M. L. et al., Resolution of a common RNA sequencing ambiguity by terminal deoxynucleotidyl transferase, Anal. Biochem., 1986, 157: 275-282.[8]Haseloff, J., Siemering, K. R., Prasher, D. C. et al., Removal of a cryptic intron and subcellular localization of green fluo-rescent protein are required to mark transgenic Arabidopsis plants brightly, Proc. Natl. Acad. Sci. USA, 1997, 94: 2122-2127.[9]Shi, B. J., Ding, S. W., Symons, R. H., Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infec-tivity of cDNA clones of tomato aspermy cucumovirus, J. Gen. Virol., 1997, 78: 1181-1185.[10]Rizzo, T. M., Palukaitis, P., Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: Genera-tion of infectious RNA transcripts, Mol. Gen. Genet., 1990, 222: 249-256.[11]Hall, R. D., The initiation and maintenance of

  10. Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants.

    Science.gov (United States)

    Dugdale, Benjamin; Mortimer, Cara L; Kato, Maiko; James, Tess A; Harding, Robert M; Dale, James L

    2014-05-01

    Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

  11. Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity.

    Science.gov (United States)

    Zeng, Yan-Syun; Gao, Ruo-Cing; Wu, Ting-Wei; Cho, Chien; Tan, Kui-Thong

    2016-08-17

    Despite the promising improvements made recently on fluorescence probes for the detection of enzymes and reactive small molecules, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag protein and three environment-sensitive fluorescence probes to overcome these two problems. The probes were constructed by following the current probe design for enzymes and reactive small molecules but with an additional benzylguanine moiety for selective SNAP-tag conjugation. The SNAP-tag conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma, whereas analyte concentrations were overestimated up to 700-fold when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically, as our probes displayed 390-fold fluorescence enhancement upon SNAP-tag conjugation, in stark contrast to the weak fluorescence of the free probes in aqueous solutions. Compared with the conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcame many key issues such as dye leakage, long preparation steps, inconsistent dye-host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

  12. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    Science.gov (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  13. Plasmon-enhanced emission from single fluorescent proteins

    Science.gov (United States)

    Donehue, Jessica E.; Haas, Beth L.; Wertz, Esther; Talicska, Courtney N.; Biteen, Julie S.

    2013-02-01

    In this work, we use evaporated gold nanoparticle films (GNPFs) as substrates for plasmon-enhanced imaging of two fluorescent proteins (FPs): mCherry and YFP. Through single-molecule epifluorescence microscopy, we show enhancement of single FP emission in the presence of GNPFs. The gold-coupled FPs demonstrate emission up to four times brighter and seven times longer lived, yielding order-of-magnitude enhancements in total photons detected. Ultimately, this results in increased localization accuracies for single-molecule imaging. Furthermore, we introduce preliminary results for enhancement of mCherry-labeled TcpP membrane proteins inside live Vibrio cholerae cells coupled to GNPFs. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

  14. Transglutamination allows production and characterization of native-sized ELPylated spider silk proteins from transgenic plants.

    Science.gov (United States)

    Weichert, Nicola; Hauptmann, Valeska; Menzel, Matthias; Schallau, Kai; Gunkel, Philip; Hertel, Thomas C; Pietzsch, Markus; Spohn, Uwe; Conrad, Udo

    2014-02-01

    In the last two decades it was shown that plants have a great potential for production of specific heterologous proteins. But high cost and inefficient downstream processing are a main technical bottleneck for the broader use of plant-based production technology especially for protein-based products, for technical use as fibres or biodegradable plastics and also for medical applications. High-performance fibres from recombinant spider silks are, therefore, a prominent example. Spiders developed rather different silk materials that are based on proteins. These spider silks show excellent properties in terms of elasticity and toughness. Natural spider silk proteins have a very high molecular weight, and it is precisely this property which is thought to give them their strength. Transgenic plants were generated to produce ELPylated recombinant spider silk derivatives. These fusion proteins were purified by Inverse Transition Cycling (ITC) and enzymatically multimerized with transglutaminase in vitro. Layers produced by casting monomers and multimers were characterized using atomic force microscopy (AFM) and AFM-based nanoindentation. The layered multimers formed by mixing lysine- and glutamine-tagged monomers were associated with the highest elastic penetration modulus. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Prediction on Antigenic Epitope Characteristics of Bt Cry2Ab Protein in Transgenic Crops

    Institute of Scientific and Technical Information of China (English)

    Jierong GAO; Ying HE; Zehong ZOU; Ailin TAO; Yuncan AI

    2012-01-01

    Abstract [Objective] This study aimed to predict the structural characteristics of Bt Cry2Ab protein in transgenic crops with bioinformatic analysis to provide the theoreti- cal clues for design of antibody Cry2Ab. [Method] The amino acid sequence of Cry2Ab protein was searched from NCBI database. The B cell epitopes were pre- dicted with DNAStar. The binding affinity between Cry2Ab protein and MHC-II molecules was analyzed with NetMHCII 2.2 Server to predict the T cell epitopes. [Result] Prediction result suggested the potential B cell epitope of Cry2Ab locating in the region of 208-215. Analysis of the binding affinity between Cry2Ab and MHC-II molecules suggested the regions of 177-185, 299-307 and 255-263 were the po- tential T cell epitopes. Human with HLA-DRB10101 alleles and HLA-DRB10701 al- leles were more sensitive to Cry2Ab protein. [Conclusion] This study facilitates to un- derstand the structural characteristics of Cry2Ab protein and provides a new clue to improve the assessment method for potential allergenicity of genetically modified food.

  16. Fluorescence energy transfer in the bi-fluorescent S-layer tandem fusion protein ECFP-SgsE-YFP.

    Science.gov (United States)

    Kainz, Birgit; Steiner, Kerstin; Sleytr, Uwe B; Pum, Dietmar; Toca-Herrera, José L

    2010-12-01

    This work reports for the first time on the fabrication of a bi-functional S-layer tandem fusion protein which is able to self-assemble on solid supports without losing its functionality. Two variants of the green fluorescent protein (GFP) were genetically combined with a self-assembly system having the remarkable opportunity to interact with each other and act as functional nanopatterning biocoating. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the cyan ECFP donor protein at the SgsE N-terminus and with the yellow YFP acceptor protein at the C-terminus. The fluorescence energy transfer was studied with spectrofluorimetry, confocal microscopy and flow cytometry, whilst protein self-assembly (on silicon dioxide particles) and structural investigations were carried out with atomic force microscopy (AFM). The fluorescence resonance energy transfer efficiency of reassembled SgsE tandem protein was 20.0 ± 6.1% which is almost the same transfer efficiency shown in solution (19.6 ± 0.1%). This work shows that bi-fluorescent S-layer fusion proteins self-assemble on silica particles retaining their fluorescent properties.

  17. Fluorescent detection of C-reactive protein using polyamide beads

    Science.gov (United States)

    Jagadeesh, Shreesha; Chen, Lu; Aitchison, Stewart

    2016-03-01

    Bacterial infection causes Sepsis which is one of the leading cause of mortality in hospitals. This infection can be quantified from blood plasma using C - reactive protein (CRP). A quick diagnosis at the patient's location through Point-of- Care (POC) testing could give doctors the confidence to prescribe antibiotics. In this paper, the development and testing of a bead-based procedure for CRP quantification is described. The size of the beads enable them to be trapped in wells without the need for magnetic methods of immobilization. Large (1.5 mm diameter) Polyamide nylon beads were used as the substrate for capturing CRP from pure analyte samples. The beads captured CRP either directly through adsorption or indirectly by having specific capture antibodies on their surface. Both methods used fluorescent imaging techniques to quantify the protein. The amount of CRP needed to give a sufficient fluorescent signal through direct capture method was found suitable for identifying bacterial causes of infection. Similarly, viral infections could be quantified by the more sensitive indirect capture method. This bead-based assay can be potentially integrated as a disposable cartridge in a POC device due to its passive nature and the small quantities needed.

  18. Cardiac Characteristics of Transgenic Mice Overexpressing Refsum Disease Gene-Associated Protein within the Heart.

    Science.gov (United States)

    Koh, J T; Choi, H H; Ahn, K Y; Kim, J U; Kim, J H; Chun, J Y; Baik, Y H; Kim, K K

    2001-09-01

    Arrhythmia is a common cardiac symptom of Refsum disease. Recently, we identified a novel neuron-specific PAHX-associated protein (PAHX-AP1), which binds to the Refsum disease gene (PAHX). In this report, we developed heart-targeted transgenic (TG) mice under the control of alpha-myosin heavy chain promoter to determine whether cardiac overexpression of PAHX-AP1 provokes cardiac involvement symptoms. Northern and in situ hybridization analyses revealed PAHX-AP1 transcript was overexpressed in TG atrium, especially in the sinoatrial node. TG mice showed tachycardia, and tachyarrhythmia was observed in 20% of TG mice. Isolated TG atria showed higher frequency beating and were more sensitive to aconitine-induced tachyarrhythmia than the wild-type, and 40% of the TG atria showed irregular beating. Action potential duration in TG atrial fiber was shortened much more than the wild-type. Systemic administration of arrhythmogenic agents induced arrhythmia in TG mice, while no arrhythmia with the same dose in nonTG mice. Our results indicate that the chronic atrial tachycardia by overexpressed neuron-specific PAHX-AP1 transgene in atrium may be responsible for the increased susceptibility to arrhythmia.

  19. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

    Directory of Open Access Journals (Sweden)

    Jinlong Guo

    2015-01-01

    Full Text Available As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV and/or Sorghum mosaic virus (SrMV, with additional differences in viral strains. RNA interference (RNAi is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms.

  20. Protein-protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection.

    Science.gov (United States)

    Benz, Christian; Retzbach, Heiko; Nagl, Stefan; Belder, Detlev

    2013-07-21

    Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of protein-protein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.

  1. Bipartite and tripartite Cucumber mosaic virus-based vectors for producing the Acidothermus cellulolyticus endo-1,4-β-glucanase and other proteins in non-transgenic plants

    Directory of Open Access Journals (Sweden)

    Hwang Min

    2012-09-01

    Full Text Available Abstract Background Using plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. The objective of this work was the development of advanced CMV-based protein production systems to produce Acidothermus cellulolyticus endo-1, 4-β-glucanase (E1 in non-transgenic plants. Results We used two new Cucumber mosaic virus (CMV-based vector systems for producing the green fluorescent protein (GFP and more importantly, the Acidothermus cellulolyticus endo-1, 4-β-glucanase (E1 in non-transgenic Nicotiana benthamiana plants. These are the inducible CMVin (CMV-based inducible and the autonomously replicating CMVar (CMV-based advanced replicating systems. We modified a binary plasmid containing the complete CMV RNA 3 cDNA to facilitate insertion of desired sequences, and to give modifications of the subgenomic mRNA 4 leader sequence yielding several variants. Quantitative RT-PCR and immunoblot analysis showed good levels of CMV RNA and coat protein accumulation for some variants of both CMVin and CMVar. When genes for E1 or GFP were inserted in place of the CMV coat protein, both were produced in plants as shown by fluorescence (GFP and immunoblot analysis. Enzymatic activity assays showed that active E1 was produced in plants with yields up to ~ 11 μg/g fresh weight (FW for specific variant constructs. We also compared in vitro CMV genomic RNA reassortants, and CMV RNA 3 mutants which lacked the C’ terminal 33 amino acids of the 3A movement protein in attempts to further increase E1 yield. Taken together specific variant constructs yielded up to ~21 μg/g FW of E1 in non-transgenic plants. Conclusions Intact, active E1 was rapidly produced in non-transgenic plants by using agroinfiltration with the CMV-based systems. This reduces the time and cost compared to that required to

  2. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    Science.gov (United States)

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins.

  3. Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.)

    Science.gov (United States)

    Tao, Geru; Shi, Tuanyuan; Tang, Xinming; Duszynski, Donald W.; Wang, Yunzhou; Li, Chao; Suo, Jingxia; Tian, Xiuling; Liu, Xianyong; Suo, Xun

    2017-01-01

    Rabbit coccidiosis causes great economic losses to world rabbitries. Little work has been done considering genetic manipulation on the etiological agents, rabbit Eimeria spp. In this study, we constructed a transgenic line of Eimeria magna (EmagER) expressing enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP) using regulatory sequences of Eimeria tenella and Toxoplasma gondii. We observed the life cycle of EmagER and confirmed that the transgenic parasites express exogenous proteins targeted to different cellular compartments throughout the entire life cycle. EYFP was expressed mainly in the nucleus and RFP both in the nucleus and cytoplasm. Then, coccidia-free, laboratory-reared 40-day-old rabbits were primarily infected with either EmagER or wild-type strain oocysts and challenged with the wild-type strain. EmagER showed similar reproductivity and immunogenicity to the wild-type strain. Finally, we examined the foreign protein-specific immune response elicited by EmagER. Rabbits were immunized with either transgenic or wild-type oocysts. Immune response against parasite-soluble antigen, EYFP and RFP in spleen, and mesenteric lymph nodes were detected by quantitative real-time PCR. The relative expression level of IFN-γ, IL-2, and TNF-α were higher in EmagER-immunized rabbits than wild-type parasites-immunized rabbits after stimulation with EYFP and RFP. Our study confirmed that a specific immune response was induced by the exogenous protein expressed by EmagER and favored future studies on application of transgenic rabbit coccidia as recombinant vaccine vectors. PMID:28167939

  4. Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.).

    Science.gov (United States)

    Tao, Geru; Shi, Tuanyuan; Tang, Xinming; Duszynski, Donald W; Wang, Yunzhou; Li, Chao; Suo, Jingxia; Tian, Xiuling; Liu, Xianyong; Suo, Xun

    2017-01-01

    Rabbit coccidiosis causes great economic losses to world rabbitries. Little work has been done considering genetic manipulation on the etiological agents, rabbit Eimeria spp. In this study, we constructed a transgenic line of Eimeria magna (EmagER) expressing enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP) using regulatory sequences of Eimeria tenella and Toxoplasma gondii. We observed the life cycle of EmagER and confirmed that the transgenic parasites express exogenous proteins targeted to different cellular compartments throughout the entire life cycle. EYFP was expressed mainly in the nucleus and RFP both in the nucleus and cytoplasm. Then, coccidia-free, laboratory-reared 40-day-old rabbits were primarily infected with either EmagER or wild-type strain oocysts and challenged with the wild-type strain. EmagER showed similar reproductivity and immunogenicity to the wild-type strain. Finally, we examined the foreign protein-specific immune response elicited by EmagER. Rabbits were immunized with either transgenic or wild-type oocysts. Immune response against parasite-soluble antigen, EYFP and RFP in spleen, and mesenteric lymph nodes were detected by quantitative real-time PCR. The relative expression level of IFN-γ, IL-2, and TNF-α were higher in EmagER-immunized rabbits than wild-type parasites-immunized rabbits after stimulation with EYFP and RFP. Our study confirmed that a specific immune response was induced by the exogenous protein expressed by EmagER and favored future studies on application of transgenic rabbit coccidia as recombinant vaccine vectors.

  5. [Progress in transgenic fish techniques and application].

    Science.gov (United States)

    Ye, Xing; Tian, Yuan-Yuan; Gao, Feng-Ying

    2011-05-01

    Transgenic technique provides a new way for fish breeding. Stable lines of growth hormone gene transfer carps, salmon and tilapia, as well as fluorescence protein gene transfer zebra fish and white cloud mountain minnow have been produced. The fast growth characteristic of GH gene transgenic fish will be of great importance to promote aquaculture production and economic efficiency. This paper summarized the progress in transgenic fish research and ecological assessments. Microinjection is still the most common used method, but often resulted in multi-site and multi-copies integration. Co-injection of transposon or meganuclease will greatly improve the efficiency of gene transfer and integration. "All fish" gene or "auto gene" should be considered to produce transgenic fish in order to eliminate misgiving on food safety and to benefit expression of the transferred gene. Environmental risk is the biggest obstacle for transgenic fish to be commercially applied. Data indicates that transgenic fish have inferior fitness compared with the traditional domestic fish. However, be-cause of the genotype-by-environment effects, it is difficult to extrapolate simple phenotypes to the complex ecological interactions that occur in nature based on the ecological consequences of the transgenic fish determined in the laboratory. It is critical to establish highly naturalized environments for acquiring reliable data that can be used to evaluate the environ-mental risk. Efficacious physical and biological containment strategies remain to be crucial approaches to ensure the safe application of transgenic fish technology.

  6. Analysis of green fluorescent protein bioluminescence in vivo and in vitro using a glow discharge

    Science.gov (United States)

    Hernández, L.; Mandujano, L. A.; Cuevas, J.; Reyes, P. G.; Osorio-González, D.

    2015-03-01

    The discovery of fluorescent proteins has been a revolution in cell biology and related sciences because of their many applications, mainly emphasizing their use as cellular markers. The green fluorescent protein (GFP) is one of the most used as it requires no cofactors to generate fluorescence and retains this property into any organism when it is expressed by recombinant DNA techniques, which is a great advantage. In this work, we analyze the emission spectra of recombinant green fluorescent protein in vivo and in vitro exposed to a glow discharge plasma of nitrogen in order to relate electron temperature to fluorescence intensity.

  7. Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein

    Directory of Open Access Journals (Sweden)

    Hadjantonakis Anna-Katerina

    2005-07-01

    Full Text Available Abstract Background DsRed the red fluorescent protein (RFP isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems. Results We have used embryonic stem cell-mediated transgenesis to evaluate mRFP1 in embryonic stem cells and mice. We find that mRFP1 exhibits the most spatially homogenous expression when compared to the native (tetrameric and variant dimeric forms of DsRed. High levels of mRFP1 expression do not affect cell morphology, developmental potential or viability and fertility of animals. High levels of widespread mRFP1 expression are maintained in a constitutive manner in embryonic stem cells in culture and in transgenic animals. We have used various optical imaging modalities to visualize mRFP1 expressing cells in culture, in embryos and adult mice. Moreover co-visualization of red, green and cyan fluorescent cells within a sample is easily achieved without the need for specialized methodologies, such as spectral deconvolution or linear unmixing. Conclusion Fluorescent proteins with excitation and/or emission profiles in the red part of the visible spectrum represent distinct partners, or longer wavelength substitutes for GFP. Not only do DsRed-based RFPs provide a genetically and spectrally distinct addition to the available repertoire of autoflorescent proteins, but by virtue of their spectral properties they

  8. Highly Fluorescent Green Fluorescent Protein Chromophore Analogues Made by Decorating the Imidazolone Ring.

    Science.gov (United States)

    Gutiérrez, Sara; Martínez-López, David; Morón, María; Sucunza, David; Sampedro, Diego; Domingo, Alberto; Salgado, Antonio; Vaquero, Juan J

    2015-12-14

    The synthesis and photophysical behavior of an unexplored family of green fluorescent protein (GFP)-like chromophore analogues is reported. The compound (Z)-4-(4-hydroxybenzylidene)-1-propyl-2-(propylamino)-1H-imidazol-5(4 H)-one (p-HBDNI, 2 a) exhibits significantly enhanced fluorescence properties relative to the parent compound (Z)-5-(4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one (p-HBDI, 1). p-HBDNI was considered as a model system and the photophysical properties of other novel 2-amino-3,5-dihydro-4H-imidazol-4-one derivatives were evaluated. Time-dependent DFT calculations were carried out to rationalize the results. The analogue AIDNI (2 c), in which the 4-hydroxybenzyl group of p-HBDNI was replaced by an azaindole group, showed improved photophysical properties and potential for cell staining. The uptake and intracellular distribution of 2 c in living cells was investigated by confocal microscopy imaging.

  9. Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Wadsworth, Jonathan D F; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Groschup, Martin H; Hope, James; Brandner, Sebastian; Asante, Emmanuel A; Collinge, John

    2013-11-01

    Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

  10. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

    Directory of Open Access Journals (Sweden)

    Mueller-Roeber Bernd

    2010-05-01

    Full Text Available Abstract Background Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL. Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols

  11. Complex Assembly Behavior During the Encapsulation of Green Fluorescent Protein Analogs in Virus Derived Protein Capsules

    NARCIS (Netherlands)

    Minten, Inge J.; Nolte, Roeland J.M.; Cornelissen, Jeroen J.L.M.

    2010-01-01

    Enzymes encapsulated in nanocontainers are a better model of the conditions inside a living cell than free enzymes in solution. In a first step toward the encapsulation of multiple enzymes inside the cowpea chlorotic mottle virus (CCMV) capsid, enhanced green fluorescent protein (EGFP) was attached

  12. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence.

    Science.gov (United States)

    Caldwell, C R

    1987-07-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.

  13. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    Science.gov (United States)

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

  14. Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.

    Science.gov (United States)

    Schoetz, Ulrike; Deliolanis, Nikolaos C; Ng, David; Pauli, Jutta; Resch-Genger, Ute; Kühn, Enrico; Heuer, Steffen; Beisker, Wolfgang; Köster, Reinhard W; Zitzelsberger, Horst; Caldwell, Randolph B

    2014-01-01

    With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.

  15. Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.

    Directory of Open Access Journals (Sweden)

    Ulrike Schoetz

    Full Text Available With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig genes by recombination, gene conversion (GC and somatic hypermutation (SHM. Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation. The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.

  16. Production of a Transgenic Mosquito Expressing Circumsporozoite Protein, a Malarial Protein, in the Salivary Gland of Anopheles stephensi (Diptera:Culicidae)

    OpenAIRE

    Matsuoka, Hiroyuki; Ikezawa, Tsunetaka; Hirai, Makoto

    2010-01-01

    We are producing a transgenic mosquito, a flying syringe, to deliver a vaccine protein to human beings via the saliva the mosquito deposits in the skin while biting. The mosquito produces a vaccine protein in the salivary gland (SG) and deposits the protein into the host's skin when it takes the host's blood. We chose circumsporozoite protein (CSP), currently the most promising malaria vaccine candidate, to be expressed in the SG of Anopheles stephensi. To transform the mosquitoes, plasmid co...

  17. Handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins in transgenic mice

    DEFF Research Database (Denmark)

    Kragh, Peter M; Pedersen, Christina B; Schmidt, Stine P;

    2007-01-01

    Abstract To investigate the in vivo handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins, three transgenic mouse lines were produced by pronuclear injection of cDNA encoding the wild-type, hSCAD-wt, and two disease causing folding variants hSCAD-319C > T and hSCAD-625G > A...

  18. Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction.

    Science.gov (United States)

    Talukder, Poulami; Chen, Shengxi; Roy, Basab; Yakovchuk, Petro; Spiering, Michelle M; Alam, Mohammad P; Madathil, Manikandadas M; Bhattacharya, Chandrabali; Benkovic, Stephen J; Hecht, Sidney M

    2015-12-29

    Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

  19. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Science.gov (United States)

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.

    2015-01-01

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  20. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Directory of Open Access Journals (Sweden)

    Bernhard Hochreiter

    2015-10-01

    Full Text Available Fluorescence- or Förster resonance energy transfer (FRET is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a cleavage; (b conformational-change; (c mechanical force and (d changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  1. Fluorescent proteins as genetically encoded FRET biosensors in life sciences.

    Science.gov (United States)

    Hochreiter, Bernhard; Garcia, Alan Pardo; Schmid, Johannes A

    2015-10-16

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  2. Tyloses and phenolic deposits in xylem vessels impede water transport in low-lignin transgenic poplars: a study by cryo-fluorescence microscopy.

    Science.gov (United States)

    Kitin, Peter; Voelker, Steven L; Meinzer, Frederick C; Beeckman, Hans; Strauss, Steven H; Lachenbruch, Barbara

    2010-10-01

    Of 14 transgenic poplar genotypes (Populus tremula × Populus alba) with antisense 4-coumarate:coenzyme A ligase that were grown in the field for 2 years, five that had substantial lignin reductions also had greatly reduced xylem-specific conductivity compared with that of control trees and those transgenic events with small reductions in lignin. For the two events with the lowest xylem lignin contents (greater than 40% reduction), we used light microscopy methods and acid fuchsin dye ascent studies to clarify what caused their reduced transport efficiency. A novel protocol involving dye stabilization and cryo-fluorescence microscopy enabled us to visualize the dye at the cellular level and to identify water-conducting pathways in the xylem. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epifluorescence microscope equipped with a cryo-stage. We could then distinguish clearly between phenolic-occluded vessels, conductive (stain-filled) vessels, and nonconductive (water- or gas-filled) vessels. Low-lignin trees contained areas of nonconductive, brown xylem with patches of collapsed cells and patches of noncollapsed cells filled with phenolics. In contrast, phenolics and nonconductive vessels were rarely observed in normal colored wood of the low-lignin events. The results of cryo-fluorescence light microscopy were supported by observations with a confocal microscope after freeze drying of cryo-planed samples. Moreover, after extraction of the phenolics, confocal microscopy revealed that many of the vessels in the nonconductive xylem were blocked with tyloses. We conclude that reduced transport efficiency of the transgenic low-lignin xylem was largely caused by blockages from tyloses and phenolic deposits within vessels rather than by xylem collapse.

  3. The electronic excited states of green fluorescent protein chromophore models

    Science.gov (United States)

    Olsen, Seth Carlton

    We explore the properties of quantum chemical approximations to the excited states of model chromophores of the green fluorescent protein of A. victoria. We calculate several low-lying states by several methods of quantum chemical calculation, including state-averaged complete active space SCF (CASSCF) methods, time dependent density functional theory (TDDFT), equation-of motion coupled cluster (EOM-CCSD) and multireference perturbation theory (MRPT). Amongst the low-lying states we identify the optically bright pipi* state of the molecules and examine its properties. We demonstrate that the state is dominated by a single configuration function. We calculate zero-time approximations to the resonance Raman spectrum of GFP chromophore models, and assign published spectra based upon these.

  4. Enhanced green fluorescent protein-mediated synthesis of biocompatible graphene.

    Science.gov (United States)

    Gurunathan, Sangiliyandi; Woong Han, Jae; Kim, Eunsu; Kwon, Deug-Nam; Park, Jin-Ki; Kim, Jin-Hoi

    2014-10-03

    Graphene is the 2D form of carbon that exists as a single layer of atoms arranged in a honeycomb lattice and has attracted great interest in the last decade in view of its physical, chemical, electrical, elastic, thermal, and biocompatible properties. The objective of this study was to synthesize an environmentally friendly and simple methodology for the preparation of graphene using a recombinant enhanced green fluorescent protein (EGFP). The successful reduction of GO to graphene was confirmed using UV-vis spectroscopy, and FT-IR. DLS and SEM were employed to demonstrate the particle size and surface morphology of GO and EGFP-rGO. The results from Raman spectroscopy suggest the removal of oxygen-containing functional groups from the surface of GO and formation of graphene with defects. The biocompatibility analysis of GO and EGFP-rGO in human embryonic kidney (HEK) 293 cells suggests that GO induces significant concentration-dependent cell toxicity in HEK cells, whereas graphene exerts no adverse effects on HEK cells even at a higher concentration (100 μg/mL). Altogether, our findings suggest that recombinant EGFP can be used as a reducing and stabilizing agent for the preparation of biocompatible graphene. The novelty and originality of this work is that it describes a safe, simple, and environmentally friendly method for the production of graphene using recombinant enhanced green fluorescent protein. Furthermore, the synthesized graphene shows excellent biocompatibility with HEK cells; therefore, biologically synthesized graphene can be used for biomedical applications. To the best of our knowledge, this is the first and novel report describing the synthesis of graphene using recombinant EGFP.

  5. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

    Science.gov (United States)

    Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

    2015-07-01

    In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.

  6. A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range.

    Science.gov (United States)

    Xue, Lin; Prifti, Efthymia; Johnsson, Kai

    2016-04-27

    We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics.

  7. Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants.

    Science.gov (United States)

    Woodard, Susan L; Mayor, Jocelyne M; Bailey, Michele R; Barker, Donna K; Love, Robert T; Lane, Jeffrey R; Delaney, Donna E; McComas-Wagner, Janet M; Mallubhotla, Hanuman D; Hood, Elizabeth E; Dangott, Lawrence J; Tichy, Shane E; Howard, John A

    2003-10-01

    Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.

  8. Bond selection in the photoisomerization reaction of anionic green fluorescent protein and kindling fluorescent protein chromophore models.

    Science.gov (United States)

    Olsen, Seth; Smith, Sean C

    2008-07-09

    The chromophores of the most widely known fluorescent proteins (FPs) are derivatives of a core p-hydroxybenzylidene-imidazolinon-5-one (HBI) motif, which usually occurs as a phenolate anion. Double bond photoisomerization of the exocyclic bridge of HBI is widely held to be an important internal conversion mechanism for FP chromophores. Herein we describe the ground and excited-state electronic structures and potential energy surfaces of two model chromophores: 4- p-hydroxybenzylidiene-1,2-dimethyl-imidazolin-5-one anion (HBDI), representing green FPs (GFPs), and 2-acetyl-4-hydroxybenylidene-1-methyl-imidazolin-5-one anion (AHBMI), representing kindling FPs (KFPs). These chromophores differ by a single substitution, but we observe qualitative differences in the potential energy surfaces which indicate inversion of bond selection in the photoisomerization reaction. Bond selection is also modulated by whether the reaction proceeds from a Z or an E conformation. These configurations correspond to fluorescent and nonfluorescent states of structurally characterized FPs, including some which can be reversibly switched by specific illumination regimes. We explain the difference in bond selectivity via substituent stabilization effects on a common set of charge-localized chemical structures. Different combinations of these structures give rise to both optically active (planar) and twisted intramolecular charge-transfer (TICT) states of the molecules. We offer a prediction of the gas-phase absorption of AHBMI, which has not yet been measured. We offer a hypothesis to explain the unusual fluorescence of AHBMI in DMF solution, as well as an experimental proposal to test our hypothesis.

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

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

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

  10. Transgenic overexpression of pregnancy-associated plasma protein-A in murine arterial smooth muscle accelerates atherosclerotic lesion development

    DEFF Research Database (Denmark)

    Conover, Cheryl A; Mason, Megan A; Bale, Laurie K

    2010-01-01

    of atherosclerotic lesions, we generated transgenic mice that express human PAPP-A in arterial smooth muscle. Four founder lines were characterized for transgenic human PAPP-A mRNA and protein expression, IGFBP-4 protease activity, and tissue specificity. In study I, apolipoprotein E knockout (ApoE KO) mice, a well...... from ApoE KO/Tg compared with ApoE KO mice (P smooth muscle of double ApoE KO/PAPP-A KO mice resulted in a 2.5-fold increase in lesion area (P = 0.002), without an effect...... on lesion number. PAPP-A transgene expression was associated with a significant increase in an IGF-responsive gene (P smooth muscle accelerates lesion progression in a mouse model...

  11. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    OpenAIRE

    Luchakivska Yu. S.; Komarnytskii I. K.; Kurchenko I. M.; Yurieva O. M.; Zhytkevich N. V.; Kuchuk M. V.

    2015-01-01

    Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the sali...

  12. A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin.

    Science.gov (United States)

    Martin, Harry; Murray, Colleen; Christeller, John; McGhie, Tony

    2008-10-01

    A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 microM (26.1 microg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 microM (approximately 0.18 microg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 microL in 384-well microplates.

  13. Bimolecular fluorescence complementation as a tool to study interactions of regulatory proteins in plant protoplasts.

    Science.gov (United States)

    Pattanaik, Sitakanta; Werkman, Joshua R; Yuan, Ling

    2011-01-01

    Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

  14. Recent progress in design of protein-based fluorescent biosensors and their cellular applications.

    Science.gov (United States)

    Tamura, Tomonori; Hamachi, Itaru

    2014-12-19

    Protein-based fluorescent biosensors have emerged as key bioanalytical tools to visualize and quantify a wide range of biological substances and events in vitro, in cells, and even in vivo. On the basis of the construction method, the protein-based fluorescent biosensors can be principally classified into two classes: (1) genetically encoded fluorescent biosensors harnessing fluorescent proteins (FPs) and (2) semisynthetic biosensors comprised of protein scaffolds and synthetic fluorophores. Recent advances in protein engineering and chemical biology not only allowed the further optimization of conventional biosensors but also facilitated the creation of novel biosensors based on unique strategies. In this review, we survey the recent studies in the development and improvement of protein-based fluorescent biosensors and highlight the successful applications to live cell and in vivo imaging. Furthermore, we provide perspectives on possible future directions of the technique.

  15. Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens

    Directory of Open Access Journals (Sweden)

    Peijnenburg Ad ACM

    2002-12-01

    Full Text Available Abstract Background Transgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm. Results Thirty-three transgenic proteins have been screened for identities of at least six contiguous amino acids shared with allergenic proteins. Twenty-two transgenic proteins showed positive results of six- or seven-contiguous amino acids length. Only a limited number of identical stretches shared by transgenic proteins (papaya ringspot virus coat protein, acetolactate synthase GH50, and glyphosate oxidoreductase and allergenic proteins could be identified as (part of potential linear epitopes. Conclusion Many transgenic proteins have identical stretches of six or seven amino acids in common with allergenic proteins. Most identical stretches are likely to be false positives. As shown in this study, identical stretches can be further screened for relevance by comparison with linear IgE-binding epitopes described in literature. In the absence of literature data on epitopes, antigenicity prediction by computer aids to select potential antibody binding sites that will need verification of IgE binding by sera binding tests. Finally, the positive outcomes of this approach warrant further clinical testing for potential allergenicity.

  16. Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens

    Science.gov (United States)

    Kleter, Gijs A; Peijnenburg, Ad ACM

    2002-01-01

    Background Transgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm. Results Thirty-three transgenic proteins have been screened for identities of at least six contiguous amino acids shared with allergenic proteins. Twenty-two transgenic proteins showed positive results of six- or seven-contiguous amino acids length. Only a limited number of identical stretches shared by transgenic proteins (papaya ringspot virus coat protein, acetolactate synthase GH50, and glyphosate oxidoreductase) and allergenic proteins could be identified as (part of) potential linear epitopes. Conclusion Many transgenic proteins have identical stretches of six or seven amino acids in common with allergenic proteins. Most identical stretches are likely to be false positives. As shown in this study, identical stretches can be further screened for relevance by comparison with linear IgE-binding epitopes described in literature. In the absence of literature data on epitopes, antigenicity prediction by computer aids to select potential antibody binding sites that will need verification of IgE binding by sera binding tests. Finally, the positive outcomes of this approach warrant further clinical testing for potential allergenicity. PMID:12477382

  17. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    Science.gov (United States)

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

  18. Transgenic proteins in agricultural biotechnology: The toxicology forum 40th annual summer meeting.

    Science.gov (United States)

    Sherman, James H; Choudhuri, Supratim; Vicini, John L

    2015-12-01

    During the 40th Annual Meeting of The Toxicology Forum, the current and potential future science, regulations, and politics of agricultural biotechnology were presented and discussed. The range of current commercial crops and commercial crop traits related to transgenic proteins were reviewed and example crop traits discussed, including insecticidal resistance conferred by Bt proteins and the development of nutritionally enhanced food such as Golden Rice. The existing regulatory framework in the USA, with an emphasis on US FDA's role in evaluating the safety of genetically engineered crops under the regulatory umbrella of the FD&C Act was reviewed. Consideration was given to the polarized politics surrounding agricultural biotechnology, the rise of open access journals, and the influence of the internet and social media in shaping public opinion. Numerous questions related to misconceptions regarding current products and regulations were discussed, highlighting the need for more scientists to take an active role in public discourse to facilitate public acceptance and adoption of new technologies and to enable science-based regulations.

  19. Cymbidium mosaic virus coat protein gene in antisense confers resistance to transgenic Nicotiana occidentalis.

    Science.gov (United States)

    Lim, S H; Ko, M K; Lee, S J; La, Y J; Kim, B D

    1999-12-31

    The nucleotide sequence of the 3'-terminal region of the Korean isolate of cymbidium mosaic virus (CyMV-Ca) from a naturally infected cattleya was determined. The sequence contains an open reading frame (ORF) coding for the viral coat protein (CP) at the 3'-end and three other ORFs (triple gene block or movement protein) of CyMV. The CP gene encodes a polypeptide chain of 220 amino acids with a molecular mass of 23,760 Da. The deduced CP sequence showed a strong homology with those of two CyMVs reported. A construct of the CyMV-Ca CP gene in the antisense orientation in the plant expression vector pMBP1 was transferred via Agrobacterium tumefaciens-mediated transformation into Nicotiana occidentalis which is a propagation host of CyMV. The T1 progeny of the transgenic plants were inoculated with CyMV and found to be highly resistant to CyMV infection.

  20. A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants.

    Science.gov (United States)

    Yamamoto, Masayuki P; Shinada, Hiroshi; Onodera, Yasuyuki; Komaki, Chihiro; Mikami, Tetsuo; Kubo, Tomohiko

    2008-06-01

    In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST-ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.

  1. A fluorescence spectroscopic study of a coagulating protein extracted from Moringa oleifera seeds.

    Science.gov (United States)

    Kwaambwa, H M; Maikokera, R

    2007-11-15

    The fluorescence studies of coagulating protein extracted from Moringa oleifera seeds have been studied using steady-state intrinsic fluorescence. The fluorescence spectra are dominated by tryptophan emission and the emission peak maximum (lambda(max)=343+ or -2nm) indicated that the tryptophan residue is not located in the hydrophobic core of the protein. Changes in solution pH affected the protein conformation as indicated by changes in the tryptophan fluorescence above pH 9 whereas the ionic strength had minimal effect. The exposure and environments of the tryptophan residue were determined using collisional quenchers.

  2. A pH-sensitive red fluorescent protein compatible with hydrophobic resin embedding

    Science.gov (United States)

    Guo, Wenyan; Gang, Yadong; Liu, Xiuli; Zhou, Hongfu; Zeng, Shaoqun

    2017-02-01

    pH sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EYFP or EGFP improved from GFP in jellyfish are good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is of urgent need. Here a pH sensitive red fluorescent protein, pHuji, is selected and verified to be compatible with hydrophobic resin embedding and thus may be promising for dual-colour chemical reactivation imaging in conjunction with EGFP or EYFP.

  3. Picosecond Fluorescence Dynamics of Tryptophan and 5-Fluorotryptophan in Monellin : Slow Water-Protein Relaxation Unmasked

    NARCIS (Netherlands)

    Xu, Jianhua; Chen, Binbin; Callis, Patrik Robert; Muiño, Pedro L; Rozeboom, Henriette J; Broos, Jaap; Toptygin, Dmitri; Brand, Ludwig; Knutson, Jay R

    2015-01-01

    Time Dependent Fluorescence Stokes (emission wavelength) Shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many fluor

  4. Picosecond Fluorescence Dynamics of Tryptophan and 5-Fluorotryptophan in Monellin : Slow Water-Protein Relaxation Unmasked

    NARCIS (Netherlands)

    Xu, Jianhua; Chen, Binbin; Callis, Patrik Robert; Muiño, Pedro L; Rozeboom, Henriette J; Broos, Jaap; Toptygin, Dmitri; Brand, Ludwig; Knutson, Jay R

    2015-01-01

    Time Dependent Fluorescence Stokes (emission wavelength) Shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many

  5. Rise-time of FRET-acceptor fluorescence tracks protein folding

    OpenAIRE

    Simon Lindhoud; Adrie H. Westphal; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Jan Willem Borst

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based F...

  6. Split green fluorescent protein as a modular binding partner for protein crystallization.

    Science.gov (United States)

    Nguyen, Hau B; Hung, Li-Wei; Yeates, Todd O; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-12-01

    A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

  7. Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation

    Directory of Open Access Journals (Sweden)

    S.S.G. Ferguson

    1998-11-01

    Full Text Available G protein-coupled receptor (GPCR activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs. Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.

  8. Alteration of Methamphetamine-induced stereotypic behaviour in transgenic mice expressing HIV-1 envelope protein gp120.

    Science.gov (United States)

    Roberts, Amanda J; Maung, Ricky; Sejbuk, Natalia E; Ake, Christopher; Kaul, Marcus

    2010-02-15

    The use of drugs for recreational purposes, in particular Methamphetamine, is associated with an increased risk of infection with human immunodeficiency virus (HIV)-1. HIV-1 infection in turn can lead to HIV-associated neurological disorders (HAND) that range from mild cognitive and motor impairment to HIV-associated dementia (HAD). Interestingly, post mortem brain specimens from HAD patients and transgenic (tg) mice expressing the viral envelope protein gp120 in the central nervous system display similar neuropathological signs. In HIV patients, the use of Methamphetamine appears to aggravate neurocognitive alterations. In the present study, we injected HIV/gp120tg mice and non-transgenic littermate control animals with Methamphetamine dissolved in Saline or Saline vehicle and assessed locomotion and stereotyped behaviour. We found that HIVgp120-transgenic mice differ significantly from non-transgenic controls in certain domains of their behavioural response to Methamphetamine. Thus this experimental model system may be useful to further study the mechanistic interaction of both the viral envelope protein and the psychostimulant drug in behavioural alterations and neurodegenerative disease.

  9. Generation of the bovine viral diarrhea virus e0 protein in transgenic astragalus and its immunogenicity in sika deer.

    Science.gov (United States)

    Gao, Yugang; Zhao, Xueliang; Zang, Pu; Liu, Qun; Wei, Gongqing; Zhang, Lianxue

    2014-01-01

    The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR), transcription was verified by reverse transcription- (RT-) PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine.

  10. Silencing of potato virus X coat protein gene in transgenic tobaccos by codon replacement that confers resistance to PVX infection

    Institute of Scientific and Technical Information of China (English)

    FENG Dejiang; LIU Xiang; MENG Kun; LIAO Lili; WEI Xiaoli; XU Honglin; ZHU Zhen

    2003-01-01

    To understand the effect of rare codon on the silencing ratio of foreign gene, some preferred codon in potato virus X (PVX) coat protein gene (cp) were substituted with synonymous rare codons. The modified PVX coat protein gene (cpm) and wild-type cp gene (cpw) were inserted into binary vector under the control of CaMV35S promoter, and these two plant expression constructs were transferred into tobacco (Nicotiana tabacum cv. Xanthi) genomes via Agrobacterium mediated method and transgenic plants were generated. Northern blot analysis of RNA isolated from these plants showed that the silencing ratio of cpm gene in transgenic tobaccos was higher than that of cpw (35% and 6.25% respectively). Run on results indicate that the silencing of cp gene happened at post-transcriptional level. The resistance of transgenic tobaccos carrying cpm genes to PVX is increased compared with that of transformants carrying cpw genes. These results suggest that the resistance of transgenic tobacco to PVX can be enhanced by codon replacement.

  11. Generation of the Bovine Viral Diarrhea Virus E0 Protein in Transgenic Astragalus and Its Immunogenicity in Sika Deer

    Directory of Open Access Journals (Sweden)

    Yugang Gao

    2014-01-01

    Full Text Available The bovine viral diarrhea virus (BVDV, a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR, transcription was verified by reverse transcription- (RT- PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine.

  12. A polarizable embedding DFT study of one-photon absorption in fluorescent proteins

    DEFF Research Database (Denmark)

    Beerepoot, Maarten; Steindal, Arnfinn H.; Kongsted, Jacob;

    2013-01-01

    A theoretical study of the one-photon absorption of five fluorescent proteins (FPs) is presented. The absorption properties are calculated using a polarizable embedding approach combined with density functional theory (PE-DFT) on the wild-type green fluorescent protein (wtGFP) and several of its...

  13. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been con...

  14. Mass spectrometry based approach for identification and characterisation of fluorescent proteins from marine organisms

    DEFF Research Database (Denmark)

    Wojdyla, Katarzyna Iwona; Rogowska-Wrzesinska, Adelina; Wrzesinski, Krzysztof

    2011-01-01

    of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples...

  15. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been...

  16. FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1

    OpenAIRE

    Raina, Shweta A.; Jeffrey Tsai; Montserrat Samsó; Fessenden, James D.

    2012-01-01

    Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) inse...

  17. Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

    LENUS (Irish Health Repository)

    Tudor, E L

    2010-05-19

    Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

  18. Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

    Science.gov (United States)

    Nienhaus, Karin; Nienhaus, G. Ulrich

    2016-11-01

    Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

  19. R26R-GR: a Cre-activable dual fluorescent protein reporter mouse.

    Directory of Open Access Journals (Sweden)

    You-Tzung Chen

    Full Text Available Green fluorescent protein (GFP and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry. This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

  20. Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

    Science.gov (United States)

    Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

  1. Fluorescence lifetime measurements of intrinsically unstructured proteins: application to α-synuclein.

    Science.gov (United States)

    Schreurs, Sarah; Kluba, Malgorzata; Meuvis, Jessika; Engelborghs, Yves

    2012-01-01

    Lifetimes of fluorescent states and their fluorescence intensities are strictly coupled and very sensitive to the environment of the fluorophores. The advantage of measuring lifetimes, next to intensities, comes from the fact that it can reveal heterogeneity and dynamic properties of this environment. In this way lifetime analysis can be used to characterize static and dynamic conformational properties and heterogeneity of fluorescent groups in different areas of a protein and as a function of time for an evolving protein. The phenomena that determine the lifetime of a label are its intrinsic properties, dynamic quenching by neighboring groups, exposure to the solvent, as well as Förster resonance energy transfer (FRET) between different groups. The basic principles of these fluorescence phenomena can be found extensively described in the excellent book of Lakowicz (Principles of fluorescence spectroscopy, 3rd edn. Springer, New York, 2006). The fluorescent groups involved are either natural amino acid side chains like tryptophan (Trp) or tyrosine (Tyr), or fluorescent labels covalently engineered into the protein. Even a single fluorescent group can show indications of heterogeneity in the local environment. If several natural fluorescent groups are present, the properties of the individual groups can be separated using site-directed mutagenesis, and additivity of their contributions can be analyzed (Engelborghs, Spectrochim Acta A Mol Biomol Spectrosc 57(11):2255-2270, 2001). If no fluorescent group is naturally present, site-directed mutagenesis can be used to introduce either a fluorescent amino acid or a cysteine allowing chemical labeling.

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

    Science.gov (United States)

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

    2014-07-01

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

  3. Changes underlying arrhythmia in the transgenic heart overexpressing Refsum disease gene-associated protein.

    Science.gov (United States)

    Koh, Jeong Tae; Jeong, Byung Chul; Kim, Jae Ha; Ahn, Young Keun; Lee, Hyang Sim; Baik, Yung Hong; Kim, Kyung Keun

    2004-01-01

    Previously, we identified a novel neuron-specific protein (PAHX-AP1) that binds to Refsum disease gene product (PAHX), and we developed transgenic (TG) mice that overexpress heart-targeted PAHX-AP1. These mice have atrial tachycardia and increased susceptibility to aconitine-induced arrhythmia. This study was undertaken to elucidate the possible changes in ion channels underlying the susceptibility to arrhythmia in these mice. RT-PCR analyses revealed that the cardiac expression of adrenergic beta(1)-receptor (ADRB1) was markedly lower, whereas voltage-gated potassium channel expression (Kv2.1) was higher in PAHX-AP1 TG mice compared with non-TG mice. However, the expression of voltage-sensitive sodium and calcium channels, and muscarinic receptor was not significantly different. Propranolol pretreatment, a non-specific beta-adrenoceptor antagonist, blocked aconitine-induced arrhythmia in non-TG mice, but not in PAHX-AP1 TG mice. Our results indicate that, in the PAHX-AP1 TG heart, the modulation of voltage-gated potassium channel and ADRB1 expression seem to be important in the electrophysiological changes associated with altered ion channel functions, but ADRB1 is not involved in the greater susceptibility to aconitine-induced arrhythmia.

  4. Mapping fast protein folding with multiple-site fluorescent probes.

    Science.gov (United States)

    Prigozhin, Maxim B; Chao, Shu-Han; Sukenik, Shahar; Pogorelov, Taras V; Gruebele, Martin

    2015-06-30

    Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.

  5. Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.

    Directory of Open Access Journals (Sweden)

    Adil Bakayan

    Full Text Available Bioluminescence recording of Ca(2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET to the green fluorescent protein (GFP. This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+ in various cell compartments. In addition, they would also serve to monitor Ca(2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA showed the highest BRET efficiency (largest energy transfer critical distance R(0 and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+ oscillations in single HeLa cells expressing tdTA. Ca(2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+ activity of HeLa cells injected subcutaneously into mice, and Ca(2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+ sensor reported to date and is, therefore, a promising probe to study Ca(2+ dynamics in whole organisms or tissues expressing the transgene.

  6. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    DEFF Research Database (Denmark)

    Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

    2001-01-01

    To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the i......To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...

  7. Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants.

    Science.gov (United States)

    Ckurshumova, Wenzislava; Caragea, Adriana E; Goldstein, Rochelle S; Berleth, Thomas

    2011-09-01

    Since the hallmark discovery of Aequorea victoria's Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants, fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types, to monitor dynamic cell fate selection processes, and to obtain cell type-specific transcriptomes. Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes. The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms. In developmental studies, the use of fluorescent proteins has become critical, where morphological markers of tissues, cell types, or differentiation stages are either not known or not easily recognizable. In this review, we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

  8. Glow in the Dark: Fluorescent Proteins as Cell and Tissue-Specific Markers in Plants

    Institute of Scientific and Technical Information of China (English)

    Wenzislava Ckurshumova; Adriana E. Caragea; Rochelle S. Goldstein; Thomas Berleth

    2011-01-01

    Since the hallmark discovery of Aequorea victoria's Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants,fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types,to monitor dynamic cell fate selection processes,and to obtain cell type-specific transcriptomes.Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes.The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms.In developmental studies,the use of fluorescent proteins has become critical,where morphological markers of tissues,cell types,or differentiation stages are either not known or not easily recognizable.In this review,we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

  9. Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

    Directory of Open Access Journals (Sweden)

    E Ferreira

    2012-07-01

    Full Text Available Transplantation of mesenchymal stem cells (MSCs with electrotransferred bone morphogenetic protein-2 (BMP-2 transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

  10. New insights in the interpretation of tryptophan fluorescence : origin of the fluorescence lifetime and characterization of a new fluorescence parameter in proteins: the emission to excitation ratio.

    Science.gov (United States)

    Albani, J R

    2007-07-01

    Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore-environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were

  11. Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence.

    Science.gov (United States)

    García-Cañas, Virginia; González, Ramón; Cifuentes, Alejandro

    2004-07-01

    The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5-0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge

  12. Effect of catalpol on senile plaques and spatial learning and memory ability in amyloid-β protein precursor/presenilin 1 double transgenic mice

    Institute of Scientific and Technical Information of China (English)

    宋冲

    2013-01-01

    Objective To investigate whether catalpol affects senile plaque formation and spatial learning and memory ability in the amyloid-βprotein precursor/presenilin 1(APP/PS1)double transgenic mice.Methods

  13. Identification of Cry1Ac and Cry2Ab proteins in transgenic cotton seeds available in Gujarat (India by ELISA method

    Directory of Open Access Journals (Sweden)

    Alka Dohare

    2014-02-01

    Full Text Available Along with the increase market of the transgenic crops, the demand for testing GMOs and for certifying non-GMO foodstuffs has increased dramatically. Within the arena of expanding techniques for identification and quantification of transgenic crops, two major approaches for detecting GMOs are still applicable on large scale, namely ELISA based protein detection and PCR based gene identification. In present study, ELISA techniques was adopted to identify the specific Cry1Ac and Cry2Ab proteins in some transgenic cotton plants seed samples viz., Gujarat cotton hybrid – 6 (BG II, Gujarat cotton hybrid – 8 (BG II and Gujarat cotton hybrid – 10 (BG II from the Gujarat state of India. The study reveals the presence of both Cry1Ac and Cry2Ab proteins in the transgenic seed samples and also demonstrated that the technique of ELISA for identification of Cry1Ac and Cry2Ab proteins is quite handy and easily adoptable.

  14. Transgene expression in the basidiomycete root pathogen Armillaria mellea.

    Science.gov (United States)

    Toward development of a genetic transformation system for Armillaria mellea, we used particle bombardment to identify promoters for driving transgene expression. The plasmid tested was pYES-hph-004iGFP, on which the green fluorescence protein gene, gfp, is linked to the Agaricus bisporus gpdII promo...

  15. Synthesis and characterization of novel 2, 2'-bipyrimidine fluorescent derivative for protein binding

    Directory of Open Access Journals (Sweden)

    Padalkar Vikas S

    2011-11-01

    Full Text Available Abstract Background Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Results Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy-[2, 2'-bipyrimidine]-4, 6-diylbis(1H-pyrazol-3, 1-diyl dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375 and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. Conclusion In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.

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

    Science.gov (United States)

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

    2012-01-01

    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.

  17. Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

    Science.gov (United States)

    Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

    2016-04-01

    The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

  18. New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

    OpenAIRE

    Kuznetsova, Alexandra A.; Kuznetsov, Nikita A.; Vorobjev, Yuri N.; Barthes, Nicolas P. F.; Benoît Y Michel; Alain Burger; Fedorova, Olga S.

    2014-01-01

    Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside...

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

    OpenAIRE

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

    2016-01-01

    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...

  20. Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations.

    Science.gov (United States)

    Smith, Elizabeth M; Hennen, Jared; Chen, Yan; Mueller, Joachim D

    2015-07-01

    This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. [Bt transgenic crops for insect-resistance and modification of Bt protein and utilization of stacking strategy].

    Science.gov (United States)

    Li, Chen; Liu, Bolin

    2015-01-01

    Insecticidal protein genes from Bacillus thuringiensis are currently the most widely used insect-resistant genes. They have been transferred to many crops for breeding and production. Among them, cotton, maize, potato and other insect-resistant crops are commercialized, creating considerable economic benefit. In this review, we summarized advances in identifying functional genes and transgenic crops for insect resistance, compared different strategies for enhancing vigor of insecticidal protein and utilizing gene stacking as well as listing valuable groups of stacked genes. In addition, the methods for multiple gene transformation was discussed.

  2. Assessment of heat shock protein 70 induction by heat in alfalfa varieties and constitutive overexpression in transgenic plants.

    Directory of Open Access Journals (Sweden)

    Nicoletta Ferradini

    Full Text Available Heat shock proteins (HSPs are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock.

  3. Development of transgenic zooplankton Artemia as a bioreactor to produce exogenous protein.

    Science.gov (United States)

    Chang, Shih-Hung; Lee, Ben-Chang; Chen, Yan-Da; Lee, Yin-Chou; Tsai, Huai-Jen

    2011-10-01

    Although the crustacean Artemia has been commonly used as an experimental organism and served as a live bait feed for aquaculture, gene transfer system on Artemia sp. to generate stable lines is not well developed. In this study, we optimized a condition for cyst-eletroporation and generated stable lines of transgenic A. sinica. Two expression plasmids directed by the hybrid promoters of cytomegalovirus (CMV) and medaka β-actin (Mβ) were co-electroporated on decapsulated cysts: pCMV-Mβ-GFP contained GFP reporter gene and pCMV-Mβ-ypGH contained yellowfin porgy GH (ypGH) cDNA. We examined the GFP shown in the Artemia larvae and found that the expression rate was 13.3% (3,219 out of 24,054 examined). We then chose 200 G0 founders which strongly expressed GFP to generate transgenic lines. Homozygotic strains derived from F4 generation of each transgenic line, A3 and A8, were obtained. We proved that transgenic lines A3 and A8 also harbored pCMV-Mβ-ypGH and produced recombinant ypGH with a concentration of 0.089 and 0.032 μg per 50 homozygotic nauplii, respectively. Ten live Artemia nauplii were fed daily to zebrafish larvae during 25 to 35 days of post-fertilization. The average body length gain rates of zebrafish larvae fed transgenic Artemia were 16-20% greater than those of control group, indicating the exogenous ypGH produced by transgenic Artemia is functional. Therefore, we concluded that the transgenesis on Artemia is developed, and transgenic Artemia might be highly potentially useful as a new bioreactor material for application in aquaculture and biological researches.

  4. Absorption, steady-state fluorescence, fluorescence lifetime, and 2D self-assembly properties of engineered fluorescent S-layer fusion proteins of Geobacillus stearothermophilus NRS 2004/3a.

    Science.gov (United States)

    Kainz, Birgit; Steiner, Kerstin; Möller, Marco; Pum, Dietmar; Schäffer, Christina; Sleytr, Uwe B; Toca-Herrera, José L

    2010-01-11

    S-layer fusion protein technology was used to design four different fluorescent fusion proteins with three different GFP mutants and the red fluorescent protein mRFP1. Their absorption spectra, steady-state fluorescence, and fluorescence lifetime were investigated as a function of pH. It was found that fluorescence intensities and lifetime of the GFP mutant S-layer fusion proteins decreased about 50% between pH 6 and pH 5. The spectral properties of the red S-layer fusion protein were minimally affected by pH variations. These results were compared with His-tagged reference fluorescent proteins, demonstrating that the S-layer protein did not change the general spectral properties of the whole fusion protein. In addition, the pK(a) values of the fluorescent S-layer fusion proteins were calculated. Finally, it was shown that the S-layer fusion proteins were able to self-assemble forming 2D nanostructures of oblique p2 symmetry with lattice parameters of about a = 11 nm, b = 14 nm, and gamma = 80 degrees . The fluorescence tag did not hinder the natural self-assembly process of the S-layer protein. The combination of the fluorescence properties and the self-assembly ability of the engineered fusion proteins make them a promising tool to generate biomimetic surfaces for future applications in nanobiotechnology at a wide range of pH.

  5. Microspectroscopic analysis of green fluorescent proteins infiltrated into mesoporous silica nanochannels.

    NARCIS (Netherlands)

    Ma, Y.; Rajendran, P.; Blum, C.; Cesa, Y.; Gartmann, N.; Bruhwiler, D.; Subramaniam, V.

    2011-01-01

    The infiltration of enhanced green fluorescent protein (EGFP) into nanochannels of different diameters in mesoporous silica particles was studied in detail by fluorescence microspectroscopy at room temperature. Silica particles from the MCM-41, ASNCs and SBA-15 families possessing nanometer-sized (3

  6. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

    Science.gov (United States)

    Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

    2014-05-01

    Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.

  7. Photo-initiated dynamics and spectroscopy of the deprotonated Green Fluorescent Protein chromophore

    DEFF Research Database (Denmark)

    Bochenkova, Anastasia; Andersen, Lars Henrik

    2013-01-01

    This chapter combines recent advances in understanding the photophysics of the chromophore anion of the Green Fluorescent Protein (GFP) from the jellyfish Aequorea Victoria. GFP and its homologues are widely used for in vivo labeling in biology through their remarkable fluorescent properties...

  8. Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli

    NARCIS (Netherlands)

    García-Cayuela, T.; Cadiñanos, de L.P.; Mohedano, M.L.; Palencia, de P.F.; Boden, D.; Wells, J.; Peláez, C.; López, P.; Requena, T.

    2012-01-01

    Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked,

  9. Green fluorescent protein (GFP) as a vital marker for studying the interaction of Phytophthora sojae and soybean

    Institute of Scientific and Technical Information of China (English)

    CHEN XiaoRen; CHENG BaoPing; WANG XinLe; DONG SuoMeng; WANG YongLin; ZHENG XiaoBo; WANG YuanChao

    2009-01-01

    Transgenic Phytophthora sojae strains that produce green fluorescent protein (GFP) were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae.The expression of GFP during different developmental stages of P.sojae was observed using fluorescent microscopy.Based on this reporter system,the histopathologic events caused by the pathogen in soybean leaves,hypocotyls and roots were monitored.Meanwhile,the difference in resistance between different soybean cultivars against P.sojae was analyzed microscopically in roots.The results indicate that GFP can be stably expressed in zoosporangia,zoospores,cysts,hyphae and oospores of .sojae.Using the GFP marker,the infecting pathogens in leaves,hypocotyls and roots of host could be distinctly visualized.The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible culUvar Hefeng 35.These results show for the first time that this eukaryotic reporter can be used in P.sojae as a stable and vital marker,allowing the study of genetics of this hemibiotrophic pathogen.

  10. New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

    Institute of Scientific and Technical Information of China (English)

    Christian Gehl; Rainer Waadt; J(o)rg Kudla; Ralf-R. Mendel; Robert Hansch

    2009-01-01

    Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluores-cence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the locali-zation of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of can-didate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the com-bination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cn×6/Cn×6 and Cn×6/Cn×7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.

  11. Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids.

    Science.gov (United States)

    Nakasu, Erich Y T; Edwards, Martin G; Fitches, Elaine; Gatehouse, John A; Gatehouse, Angharad M R

    2014-01-01

    Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.

  12. Transgenic plants expressing -ACTX-Hv1a and snowdrop lectin (GNA fusion protein show enhanced resistance to aphids

    Directory of Open Access Journals (Sweden)

    Erich Y.T. Nakasu

    2014-11-01

    Full Text Available Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin w-ACTX-Hv1a linked to snowdrop lectin (GNA was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6±4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after seven days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50=0.73 mg/ml after two days against LC50=1.81 mg/ml for M. persicae, as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.

  13. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

    Directory of Open Access Journals (Sweden)

    Yoko Hayashi-Takanaka

    Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

  14. Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology.

    Science.gov (United States)

    Ah-Fong, Audrey M V; Judelson, Howard S

    2011-09-01

    Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cassette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and peroxisomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  15. Very bright green fluorescent proteins from the Pontellid copepod Pontella mimocerami.

    Directory of Open Access Journals (Sweden)

    Marguerite E Hunt

    Full Text Available BACKGROUND: Fluorescent proteins (FP homologous to the green fluorescent protein (GFP from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development. RESULTS: Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae, which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M(-1 cm(- and quantum yields (0.92, which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness. CONCLUSIONS: The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.

  16. Bright fluorescence monitoring system utilizing Zoanthus sp. green fluorescent protein (ZsGreen for human G-protein-coupled receptor signaling in microbial yeast cells.

    Directory of Open Access Journals (Sweden)

    Yasuyuki Nakamura

    Full Text Available G-protein-coupled receptors (GPCRs are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer's disease and Parkinson's disease, respectively were chosen as human GPCR(s. The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s.

  17. Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

    National Research Council Canada - National Science Library

    Danielle E. Johnson; Hui-wang Ai; Peter Wong; James D. Young; Robert E. Campbell; Joseph R. Casey

    2009-01-01

    .... We describe a new approach to monitor H + /uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here...

  18. Fiber-optic system for monitoring fast photoactivation dynamics of optical highlighter fluorescent proteins.

    Science.gov (United States)

    Pei, Zhiguo; Qin, Lingsong; Zhang, Zhihong; Zeng, Shaoqun; Huang, Zhen-Li

    2011-08-01

    Characterizing the photoactivation performance of optical highlighter fluorescent proteins is crucial to the realization of photoactivation localization microscopy. In contrast to those fluorescence-based approaches that require complex data processing and calibration procedures, here we report a simple and quantitative alternative, which relies on the measurement of small absorption spectra changes over time with a fiber-optic system. Using Dronpa as a representative highlighter protein, we have investigated the capacity of this system in monitoring the fast photoactivation process.

  19. [Transgenic animals bioreactors].

    Science.gov (United States)

    Gou, Ke-Mian; An, Xiao-Rong; Tian, Jian-Hui; Chen, Yong-Fu

    2002-01-01

    The production of human recombinant proteins in milk of transgenic farm animals offers a safe, very cost-effective source of commercially important proteins that cannot be produced as efficiently in adequate quantities by other methods. This review has summarized the current status of gene selection, vector construct, transgenic methods, economics, and obvious potential in transgenic animals bioreactors. Recently, a more powerful approach was adopted in the transgenic animals founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic farm animals, product consistency, and time of product development. The successful adaptation of Cre-/lox P-mediated site-specific DNA recombination systems in farm animals will offer unprecedented possibilities for generating transgenic animals.

  20. Neoplastic transformation of T lymphocytes through transgenic expression of a virus host modification protein.

    Directory of Open Access Journals (Sweden)

    Sílvia Cristina Paiva Almeida

    Full Text Available Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4(+CD8(+CD69(- lymphoma. The CD4(+CD8(+CD69(- cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4(+CD8(+CD69(- thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4(+CD8(+ CD69(- thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.

  1. Capillary electrophoresis coupled to fluorescence spectroscopy for protein characterisation

    NARCIS (Netherlands)

    de Kort, B.J.

    2012-01-01

    Proteins are essential molecules in all living organisms. Their involvement in numerous biological processes has led to the development of protein-based medicines (biopharmaceuticals). For good understanding of the properties and function of endogenous proteins and biopharmaceuticals, extensive prot

  2. [Spectral diversity among the members of the family of Green Fluorescent Protein in hydroid jellyfish (Cnidaria, Hydrozoa)].

    Science.gov (United States)

    Ianushevich, Iu G; Shagin, D A; Fradkov, A F; Shakhbazov, K S; Barsova, E V; Gurskaia, N G; Labas, Iu A; Matts, M V; Luk'ianov, k A; Lul'ianov, S A

    2005-01-01

    The cDNAs encoding the genes of new proteins homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria were cloned. Two green fluorescent proteins from one un-identified anthojellyfish, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthojellyfish were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.

  3. [Advances in safety studies of soil Bt toxin proteins released from transgenic Bt crops].

    Science.gov (United States)

    Bai, Yaoyu; Jiang, Mingxing; Cheng, Jia; Jiang, Yonghou

    2003-11-01

    Commercialized transgenic Bt (Bacillus thuringiensis) crops are permitted for field growth in a large scale, which leads to significant issues of ecological risk assessment in soil ecosystem. In this paper, some general safety problems involving in the soil Bt active toxins released from insect-resistant transgenic Bt crops in the forms of plant residues, root exudates and pollens were reviewed, including their adsorption by soil active-particles, their insecticidal activity, persistence, and biodegradation by soil microbes, and their effects on soil organisms.

  4. Development of dipsticks for simultaneous detection of vip3A and cry1Ab/cry1Ac transgenic proteins.

    Science.gov (United States)

    Kumar, Rajesh

    2012-01-01

    The number of genetically modified (GM) crops being cultivated and its produce reaching market is increasing every year. The transgenes (vip3A, cry1Ab, and cry1Ac) from Bacillus thuringiensis are being used by plant breeders, apart from other transgenes for developing insect pest-resistant GM crops. It is therefore necessary to develop an easy, rapid, and reliable detection assay to discriminate GM crops and non-GM crops. Dipstick strips using colloidal gold-labeled polyclonal antibodies were developed for simultaneous detection of Vip3A and Cry1Ab/CrylAc proteins. The assay was essentially based on the sandwich format of immunoassay, which was completed within 10 min, and the results were evaluated visually. The detection limits were 50 ng/mL (50 ppb) for both CrylAc and CrylAb proteins, and 100 ng/mL (100 ppb) for Vip3A protein. The developed dipsticks are suitable for on-site simultaneous screening of GM crops bearing two proteins, which, in turn, reduce cost and time of the assay.

  5. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    DEFF Research Database (Denmark)

    Hojman, Pernille; Eriksen, Jens; Gehl, Julie

    2009-01-01

    DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly...... weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability....

  6. Heat generation and light scattering of green fluorescent protein-like pigments in coral tissue.

    Science.gov (United States)

    Lyndby, Niclas H; Kühl, Michael; Wangpraseurt, Daniel

    2016-05-26

    Green fluorescent protein (GFP)-like pigments have been proposed to have beneficial effects on coral photobiology. Here, we investigated the relationships between green fluorescence, coral heating and tissue optics for the massive coral Dipsastraea sp. (previously Favia sp.). We used microsensors to measure tissue scalar irradiance and temperature along with hyperspectral imaging and combined imaging of variable chlorophyll fluorescence and green fluorescence. Green fluorescence correlated positively with coral heating and scalar irradiance enhancement at the tissue surface. Coral tissue heating saturated for maximal levels of green fluorescence. The action spectrum of coral surface heating revealed that heating was highest under red (peaking at 680 nm) irradiance. Scalar irradiance enhancement in coral tissue was highest when illuminated with blue light, but up to 62% (for the case of highest green fluorescence) of this photon enhancement was due to green fluorescence emission. We suggest that GFP-like pigments scatter the incident radiation, which enhances light absorption and heating of the coral. However, heating saturates, because intense light scattering reduces the vertical penetration depth through the tissue eventually leading to reduced light absorption at high fluorescent pigment density. We conclude that fluorescent pigments can have a central role in modulating coral light absorption and heating.

  7. Expression of Helicobacter pylori TonB protein in transgenic Arabidopsis thaliana: toward production of vaccine antigens in plants.

    Science.gov (United States)

    Kalbina, Irina; Engstrand, Lars; Andersson, Sören; Strid, Ake

    2010-10-01

    The aim of this study was to produce a recombinant version of the highly antigenic Helicobacter pylori TonB (iron-dependent siderophore transporter protein HP1341) in transgenic plants as a candidate oral vaccine antigen. Using Agrobacterium-mediated gene transfer, we introduced three different constructs of the tonB gene into the genome of the model plant Arabidopsis thaliana. We investigated transgene insertion by PCR, produced TonB antibodies for analysis of the production of the recombinant protein in plants, verified the identity of the protein produced by mass spectrometry analysis, and analyzed the number of genetic inserts in the plants by Southern blotting. Three different constructs of the expression cassette (full-length tonB, tonB truncated in the 5' end removing the codons for a transmembrane helix, and the latter construct with codons for the endoplasmic reticulum SEKDEL retention signal added to the 3' end) were used to find the most effective way to express the TonB antigen. Production of TonB protein was detected in plants transformed with each of the constructs, confirmed by both Western blotting and mass spectrometry analysis. No considerable differences in protein expression from the three different constructs were observed. The protein concentration in the plants was at least 0.05% of the total soluble proteins. The Helicobacter pylori TonB protein can be produced in Arabidopsis thaliana plants in a form that is recognizable by rabbit anti-TonB antiserum. These TonB-expressing plants are highly suitable for animal studies of oral administration as a route for immunization against Helicobacter infections. © 2010 Blackwell Publishing Ltd.

  8. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.

    Directory of Open Access Journals (Sweden)

    Michele L Markwardt

    Full Text Available Cyan fluorescent proteins (CFPs, such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

  9. Prox1 expression in the endolymphatic sac revealed by whole-mount fluorescent imaging of Prox1-GFP transgenic mice.

    Science.gov (United States)

    Miyashita, Takenori; Burford, James L; Hong, Young-Kwon; Gevorgyan, Haykanush; Lam, Lisa; Hoshikawa, Hiroshi; Mori, Nozomu; Peti-Peterdi, Janos

    2015-01-30

    This study describes a technical breakthrough in endolymphatic sac research, made possible by the use of the recently generated Prox1-GFP transgenic mouse model. Whole-mount imaging techniques through the decalcified temporal bone and three-dimensional observations of Prox1-GFP mouse tissue revealed the positive labeling of the endolymphatic sac in adult stage, and allowed, for the first time, the GFP-based identification of endolymphatic sac epithelial cells. Prox1 expression was observed in all parts of the endolymphatic sac epithelia. In intermediate portion of the endolymphatic sac, mitochondria-rich cells did not express Prox1, although ribosome-rich cells showed strong GFP labeling. The anatomical relationship between the endolymphatic sac and the surrounding vasculature was directly observed. In the endolymphatic sac, expression of Prox1 may suggest progenitor cell-like pluripotency or developmental similarity to systemic lymphatic vessels in other organs. This whole-mount imaging technique of the endolymphatic sac can be combined with other conventional histological, sectioning, and labeling techniques and will be very useful for future endolymphatic sac research.

  10. OsSGL, a Novel DUF1645 Domain-Containing Protein, Confers Enhanced Drought Tolerance in Transgenic Rice and Arabidopsis.

    Science.gov (United States)

    Cui, Yanchun; Wang, Manling; Zhou, Huina; Li, Mingjuan; Huang, Lifang; Yin, Xuming; Zhao, Guoqiang; Lin, Fucheng; Xia, Xinjie; Xu, Guoyun

    2016-01-01

    Drought is a major environmental factor that limits plant growth and crop productivity. Genetic engineering is an effective approach to improve drought tolerance in various crops, including rice (Oryza sativa). Functional characterization of relevant genes is a prerequisite when identifying candidates for such improvements. We investigated OsSGL (Oryza sativa Stress tolerance and Grain Length), a novel DUF1645 domain-containing protein from rice. OsSGL was up-regulated by multiple stresses and localized to the nucleus. Transgenic plants over-expressing or hetero-expressing OsSGL conferred significantly improved drought tolerance in transgenic rice and Arabidopsis thaliana, respectively. The overexpressing plants accumulated higher levels of proline and soluble sugars but lower malondialdehyde (MDA) contents under osmotic stress. Our RNA-sequencing data demonstrated that several stress-responsive genes were significantly altered in transgenic rice plants. We unexpectedly observed that those overexpressing rice plants also had extensive root systems, perhaps due to the altered transcript levels of auxin- and cytokinin-associated genes. These results suggest that the mechanism by which OsSGL confers enhanced drought tolerance is due to the modulated expression of stress-responsive genes, higher accumulations of osmolytes, and enlarged root systems.

  11. Transgenic Expression of a Functional Fragment of Harpin Protein Hpa1 in Wheat Represses English Grain Aphid Infestation

    Institute of Scientific and Technical Information of China (English)

    XU Man-yu; ZHOU Ting; ZHAO Yan-ying; LI Jia-bao; XU Heng; DONG Han-song; ZHANG Chun-ling

    2014-01-01

    The harpin protein Hpa1 produced by the rice bacterial blight pathogen promotes plant growth and induces plant resistance to pathogens and insect pests. The region of 10-42 residues (Hpa110-42) in the Hpa1 sequence is critical as the isolated Hpa110-42 fragment is 1.3-7.5-fold more effective than the full length in inducing plant growth and resistance. Here we report that transgenic expression of Hpa110-42 in wheat induces resistance to English grain aphid, a dominant species of wheat aphids. Hpa110-42-induced resistance is effective to inhibit the aphid behavior in plant preference at the initial colonization stage and repress aphid performances in the reproduction, nymph growth, and instar development on transgenic plants. The resistance characters are correlated with enhanced expression of defense-regulatory genes (EIN2, PP2-A, and GSL10) and consistent with induced expression of defense response genes (Hel, PDF1.2, PR-1b, and PR-2b). As a result, aphid infestations are alleviated in transgenic plants. The level of Hpa110-42-induced resistance in regard to repression of aphid infestations is equivalent to the effect of chemical control provided by an insecticide. These results suggested that the defensive role of Hpa110-42 can be integrated into breeding germplasm of the agriculturally signiifcant crop with a great potential of the agricultural application.

  12. High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

    Directory of Open Access Journals (Sweden)

    Amarjeet Kumar Singh

    Full Text Available Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp, which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

  13. Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

    Science.gov (United States)

    Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

    2016-02-01

    Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.

  14. Fluorescent Pressure Response of Protein-Nanocluster Polymer Composites

    Science.gov (United States)

    2016-05-01

    fluorescence spectra of BSA:AuNCs found in literature.10 Notably, the BSA:AuNCs experience peak broadening and blue shifting upon incorporation into the...semiconductor quantum dots. Nature Biotechnology . 2004;22:969–976. 9. Medintz IL, Uyeda TH, Goldman ER, Mattoussi H. Quantum dot bioconjugates for imaging

  15. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    Administrator

    times faster in E. coli and exhibits eighteen-fold greater fluorescence .... The generation time (g, min) necessary to double the .... Fitted models for µg GFPuv/ mL adjusted from the polynomial model (µg GFPuv/ mL = 10.28 - 1.13 x1 - 5.65 x3 +.

  16. Recloned transgenic pigs possess normal reproductive performance and stable genetic transmission capacity.

    Science.gov (United States)

    Cao, Zubing; Li, Yan; Wen, Xiao; Li, Zhiyuan; Mi, Changsheng; Zhang, Zaihu; Li, Ning; Li, Qiuyan

    2014-02-01

    The present study investigated whether a recloning procedure would affect the reproductive performance or the germline transmission capacity of recloned transgenic pigs. This study has also laid the foundation for the development of elite transgenic swine breeds in the future. Recloned transgenic pigs were developed from ear tissue fibroblasts of primary transgenic cloned pigs using a recloning procedure, and their reproductive performance and exogenous gene transmission were analyzed. Two transgenic cell lines with different genetic backgrounds (derived from a female miniature pig and a male Landrace pig) with stable expression of green fluorescent protein (GFP) were established successfully. Furthermore, recloned transgenic embryos were developed to full term successfully. One female Chinese experimental miniature piglet (CEMP) (GFP+) and three male Landrace piglets (GFP+) were delivered naturally. Furthermore, the index values for the reproductive characteristics of the recloned transgenic pigs, such as puberty, gestation period, sperm volume and sperm concentration, were not significantly different from those of conventionally bred pigs. In addition, 53% of the F1 offspring of the recloned transgenic pigs were GFP positive. These results demonstrate that ear tissue fibroblasts from primary transgenic cloned pigs efficiently support the full-term development of recloned transgenic embryos. Furthermore, recloned transgenic pigs maintain normal reproductive performance and stable germline (genetic) transmission capacities.

  17. Fluorescent Ensemble Based on Bispyrene Fluorophore and Surfactant Assemblies: Sensing and Discriminating Proteins in Aqueous Solution.

    Science.gov (United States)

    Fan, Junmei; Ding, Liping; Bo, Yu; Fang, Yu

    2015-10-14

    A particular bispyrene fluorophore (1) with two pyrene moieties covalently linked via a hydrophilic spacer was synthesized. Fluorescence measurements reveal that the fluorescence emission of 1 could be well modulated by a cationic surfactant, dodecyltrimethylammonium bromide (DTAB). Protein sensing studies illustrate that the selected ensemble based on 1/DTAB assemblies exhibits ratiometric responses to nonmetalloproteins and turn-off responses to metalloproteins, which can be used to differentiate the two types of proteins. Moreover, negatively charged nonmetalloproteins can be discriminated from the positively charged ones according to the difference in ratiometric responses. Fluorescence sensing studies with control bispyrenes indicate that the polarity of the spacer connecting two pyrene moieties plays an important role in locating bispyrene fluorophore in DTAB assemblies, which further influences its sensing behaviors to noncovalent interacting proteins. This study sheds light on the influence of the probe structure on the sensing performance of a fluorescent ensemble based on probe and surfactant assemblies.

  18. Production of transgenic calves by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; WAN Rong; HUANG Yinghua; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Lili; WANG Haiping; TANG Bo; LIU Ying; LI Rong

    2004-01-01

    Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

  19. Effect of pH on the Heat-Induced Denaturation and Renaturation of Green Fluorescent Protein: A Laboratory Experiment

    Science.gov (United States)

    Flores, Rosa V.; Sola, Hilda M.; Torres, Juan C.; Torres, Rafael E.; Guzman, Ernick E.

    2013-01-01

    A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence,…

  20. Effect of pH on the Heat-Induced Denaturation and Renaturation of Green Fluorescent Protein: A Laboratory Experiment

    Science.gov (United States)

    Flores, Rosa V.; Sola, Hilda M.; Torres, Juan C.; Torres, Rafael E.; Guzman, Ernick E.

    2013-01-01

    A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence,…

  1. Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

    Science.gov (United States)

    Ast, Cindy; Foret, Jessica; Oltrogge, Luke M; De Michele, Roberto; Kleist, Thomas J; Ho, Cheng-Hsun; Frommer, Wolf B

    2017-09-05

    Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

  2. Receptor-associated protein (RAP plays a central role in modulating Abeta deposition in APP/PS1 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Guilian Xu

    Full Text Available BACKGROUND: Receptor associated protein (RAP functions in the endoplasmic reticulum (ER to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP and lipoprotein receptor 11 (SorLA/LR11. Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP, including processes involved in the generation, catabolism and deposition of beta-amyloid (Abeta peptides. METHODOLOGY/PRINCIPAL FINDINGS: Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9 were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (p<0.05]. Changes in the levels of these proteins in the brains of [APPswe/PS1dE9](+/-/RAP(+/- mice correlated with 30-40% increases in amyloid deposition by 9 months of age. CONCLUSIONS/SIGNIFICANCE: Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Abeta catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.

  3. Redox Proteomic Profiling of Specifically Carbonylated Proteins in the Serum of Triple Transgenic Alzheimer’s Disease Mice

    Directory of Open Access Journals (Sweden)

    Liming Shen

    2016-04-01

    Full Text Available Oxidative stress is a key event in the onset and progression of neurodegenerative diseases, including Alzheimer’s disease (AD. To investigate the role of oxidative stress in AD and to search for potential biomarkers in peripheral blood, serums were collected in this study from the 3-, 6-, and 12-month-old triple transgenic AD mice (3×Tg-AD mice and the age- and sex-matched non-transgenic (non-Tg littermates. The serum oxidized proteins were quantified by slot-blot analysis and enzyme-linked immunosorbent assay (ELISA to investigate the total levels of serum protein carbonyl groups. Western blotting, in conjunction with two-dimensional gel electrophoresis (2D-Oxyblot, was employed to identify and quantify the specifically-carbonylated proteins in the serum of 3×Tg-AD mice. The results showed that the levels of serum protein carbonyls were increased in the three month old 3×Tg-AD mice compared with the non-Tg control mice, whereas no significant differences were observed in the six and 12 months old AD mice, suggesting that oxidative stress is an early event in AD progression. With the application of 2D-Oxyblot analysis, (immunoglobin Ig gamma-2B chain C region (IGH-3, Ig lambda-2 chain C region (IGLC2, Ig kappa chain C region (IGKC, and Ig kappa chain V-V region HP R16.7 were identified as significantly oxidized proteins compared with the control. Among them IGH-3 and IGKC were validated via immunoprecipitation and Western blot analysis. Identification of oxidized proteins in the serums of 3×Tg-AD mice can not only reveal potential roles of those proteins in the pathogenesis of AD but also provide potential biomarkers of AD at the early stage.

  4. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation

    OpenAIRE

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-01-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21–22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, di...

  5. Transfer of Bt-toxin protein gene into maize by high-velocity microprojectile bombardments and regeneration of transgenic plants

    Institute of Scientific and Technical Information of China (English)

    王国英; 杜天兵; 张宏; 谢友菊; 戴景瑞; 米景九; 李太源; 田颖川; 乔利亚; 莽克强

    1995-01-01

    Bt-toxin protein gene was successfully transferred into maize by the microprojectile bombard-ments of cell suspension,embryogenic calli and immature embryos with a Chinese-made particle gun(JQ-700).Although the bombarded embryogenic calli and immature embryos produced less mean transformants per dishthan the cell suspensions,they were the suitable materials for maize transformation because their culture andregeneration have been achieved in most maize cultivars.The evaluation on the resistance of transgenic plantsto corn borer shows the significant difference between them,from highly resistant to susceptible.

  6. Two-plasmid vector system for independently controlled expression of green and red fluorescent fusion proteins in Staphylococcus aureus.

    Science.gov (United States)

    Brzoska, Anthony J; Firth, Neville

    2013-05-01

    We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

  7. Transgenic soya bean seeds accumulating β-carotene exhibit the collateral enhancements of oleate and protein content traits.

    Science.gov (United States)

    Schmidt, Monica A; Parrott, Wayne A; Hildebrand, David F; Berg, R Howard; Cooksey, Amanda; Pendarvis, Ken; He, Yonghua; McCarthy, Fiona; Herman, Eliot M

    2015-05-01

    Transgenic soya bean (Glycine max) plants overexpressing a seed-specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 μg β carotene g(-1) dry seed weight with a desirable 12:1 ratio of β to α. The β carotene accumulating seeds exhibited a shift in oil composition increasing oleic acid with a concomitant decrease in linoleic acid and an increase in seed protein content by at least 4% (w/w). Elevated β-carotene accumulating soya bean cotyledons contain 40% the amount of abscisic acid compared to nontransgenic cotyledons. Proteomic and nontargeted metabolomic analysis of the mid-maturation β-carotene cotyledons compared to the nontransgenic did not reveal any significant differences that would account for the altered phenotypes of both elevated oleate and protein content. Transcriptomic analysis, confirmed by RT-PCR, revealed a number of significant differences in ABA-responsive transcripton factor gene expression in the crtB transgenics compared to nontransgenic cotyledons of the same maturation stage. The altered seed composition traits seem to be attributed to altered ABA hormone levels varying transcription factor expression. The elevated β-carotene, oleic acid and protein traits in the β-carotene soya beans confer a substantial additive nutritional quality to soya beans. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  8. Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

    Directory of Open Access Journals (Sweden)

    Kuang Lin-Yun

    2008-10-01

    Full Text Available Abstract Background The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed. Results We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP, and prey proteins are tagged with the N-terminal portions of either Venus (nVenus or Cerulean (nCerulean. Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin α-1 in the cytoplasm, whereas interaction of VirE2 with a different importin α isoform, importin α-4, occurs predominantly in the nucleus. Conclusion Multi

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

    Science.gov (United States)

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

    2014-05-01

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

  10. Improved Fluorescent Protein Contrast and Discrimination by Optically Controlling Dark State Lifetimes.

    Science.gov (United States)

    Chen, Yen-Cheng; Dickson, Robert M

    2017-02-16

    Modulation and optical control of photoswitchable fluorescent protein (PS-FP) dark state lifetimes drastically improves sensitivity and selectivity in fluorescence imaging. The dark state population of PS-FPs generates an out-of-phase fluorescence component relative to the sinusoidally modulated 488 nm laser excitation. Because this apparent phase advanced emission results from slow recovery to the fluorescent manifold, we hasten recovery and, therefore, modulation frequency by varying coillumination intensity at 405 nm. As 405 nm illumination regenerates the fluorescent ground state more rapidly than via thermal recovery, we experimentally demonstrate that secondary illumination can control PS-FPs dark state lifetime to act as an additional dimension for discriminating spatially and spectrally overlapping emitters. This experimental combination of out of phase imaging after optical modulation (OPIOM) and synchronously amplified fluorescence image recovery (SAFIRe) optically controls the fluorescent protein dark state lifetimes for improved time resolution, with the resulting modulation-based selective signal recovery being quantitatively modeled. The combined experimental results and quantitative numerical simulations further demonstrate the potential of SAFIRe-OPIOM for wide-field biological imaging with improved speed, sensitivity, and optical resolution over other modulation-based fluorescence microscopies.

  11. Illuminating plant biology: using fluorescent proteins for high-throughput analysis of protein localization and function in plants.

    Science.gov (United States)

    DeBlasio, Stacy L; Sylvester, Anne W; Jackson, David

    2010-03-01

    First discovered in jellyfish, fluorescent proteins (FPs) have been successfully optimized for use as effective biomarkers within living plant cells. When exposed to light, FPs fused to a protein or regulatory element will fluoresce, and non-invasively mark expression and protein localization, which allows for the in vivo monitoring of diverse cellular processes. In this review, we discuss how FP technology has evolved from small-scale analysis of individual genes to more high-throughput techniques for global expression and functional profiling in plants.

  12. Lumazine Protein from the Bioluminescent Bacterium Photobacterium Phosphoreum: A Fluorescence Study of the Protein-Ligand Equilibrium

    NARCIS (Netherlands)

    Visser, A.J.W.G.; Lee, J.

    1980-01-01

    The changes of fluorescence spectral distribution, polarization, and lifetime of the lumazine protein from Photobacterium phosphoreum can be interpreted in terms of an equilibrium between the protein and its dissociated prosthetic group 6,7-dimethyl-8-(1′-D-ribityl)lumazine. The equilibrium is

  13. Lumazine Protein from the Bioluminescent Bacterium Photobacterium Phosphoreum: A Fluorescence Study of the Protein-Ligand Equilibrium

    NARCIS (Netherlands)

    Visser, A.J.W.G.; Lee, J.

    1980-01-01

    The changes of fluorescence spectral distribution, polarization, and lifetime of the lumazine protein from Photobacterium phosphoreum can be interpreted in terms of an equilibrium between the protein and its dissociated prosthetic group 6,7-dimethyl-8-(1′-D-ribityl)lumazine. The equilibrium is rapid

  14. Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C

    DEFF Research Database (Denmark)

    Kristensen, B; Birkelund, Svend; Jørgensen, PL

    2003-01-01

    . Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation......Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent...... along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein...

  15. Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

    OpenAIRE

    2015-01-01

    Currently available methodologies for measuring protein synthesis rates rely on metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides. These approaches are hampered by several limitations and cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a novel method for monitoring protein synthesis in specific cells and tissues of live Caenorhabditis elegans animals. Fluorescent reporter proteins such as...

  16. PNA-induced assembly of fluorescent proteins using DNA as a framework

    OpenAIRE

    2013-01-01

    Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein−PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)−peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation ...

  17. Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells.

    Science.gov (United States)

    Lubbeck, Jennifer L; Dean, Kevin M; Ma, Hairong; Palmer, Amy E; Jimenez, Ralph

    2012-05-01

    Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm(2)). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S(beam3)/S(beam1)) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.

  18. Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2015-10-01

    Full Text Available The application of fluorescent proteins as expression markers and protein fusion partners has provedimmensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 arecommonly fluorescent marker protein which is used for biotechnology and cell biology research. The presentstudy was designed to identify the expression vector that suitable to ligate with DNA encoding HBV coreprotein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We alsocompared and quantified the expressed fluorescent protein which predominantly localized in the cellcompartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study ofthan EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest,the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a muchhigher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localizationthan DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescencewhen excited by blue light, without any exogenously added substrate or cofactor, events inside living cell canthus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shownthat EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study intoHuH-7 cell.Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization

  19. Measurement of protein-like fluorescence in river and waste water using a handheld spectrophotometer.

    Science.gov (United States)

    Baker, Andy; Ward, David; Lieten, Shakti H; Periera, Ryan; Simpson, Ellie C; Slater, Malcolm

    2004-07-01

    Protein-like fluorescence intensity in rivers increases with increasing anthropogenic DOM inputs from sewerage and farm wastes. Here, a portable luminescence spectrophotometer was used to investigate if this technology could be used to provide both field scientists with a rapid pollution monitoring tool and process control engineers with a portable waste water monitoring device, through the measurement of river and waste water tryptophan-like fluorescence from a range of rivers in NE England and from effluents from within two waste water treatment plants. The portable spectrophotometer determined that waste waters and sewerage effluents had the highest tryptophan-like fluorescence intensity, urban streams had an intermediate tryptophan-like fluorescence intensity, and the upstream river samples of good water quality the lowest tryptophan-like fluorescence intensity. Replicate samples demonstrated that fluorescence intensity is reproducible to +/- 20% for low fluorescence, 'clean' river water samples and +/- 5% for urban water and waste waters. Correlations between fluorescence measured by the portable spectrophotometer with a conventional bench machine were 0.91; (Spearman's rho, n = 143), demonstrating that the portable spectrophotometer does correlate with tryptophan-like fluorescence intensity measured using the bench spectrophotometer.

  20. Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

    Science.gov (United States)

    Krishna, K Vijaya; Ghosh, Subhadip; Sharma, Bikramjit; Singh, Leeju; Mukherjee, Saptarshi; Verma, Sandeep

    2015-11-24

    Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

  1. Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

    Directory of Open Access Journals (Sweden)

    Siepert Eva-Maria

    2012-09-01

    Full Text Available Abstract Background Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP. Results As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv Ki-4(scFv and the anti-MucI single-chain fragment variable M12(scFv. During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal

  2. Receptor-associated protein (RAP) plays a central role in modulating Abeta deposition in APP/PS1 transgenic mice.

    Science.gov (United States)

    Xu, Guilian; Karch, Celeste; Li, Ning; Lin, Nianwei; Fromholt, David; Gonzales, Victoria; Borchelt, David R

    2008-09-08

    Receptor associated protein (RAP) functions in the endoplasmic reticulum (ER) to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP) and lipoprotein receptor 11 (SorLA/LR11). Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP), including processes involved in the generation, catabolism and deposition of beta-amyloid (Abeta) peptides. Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (pRAP(+/-) mice correlated with 30-40% increases in amyloid deposition by 9 months of age. Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Abeta catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.

  3. Analysis of Quality-Related Parameters in Mature Kernels of Polygalacturonase Inhibiting Protein (PGIP) Transgenic Bread Wheat Infected with Fusarium graminearum.

    Science.gov (United States)

    Masci, Stefania; Laino, Paolo; Janni, Michela; Botticella, Ermelinda; Di Carli, Mariasole; Benvenuto, Eugenio; Danieli, Pier Paolo; Lilley, Kathryn S; Lafiandra, Domenico; D'Ovidio, Renato

    2015-04-22

    Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters.

  4. Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells.

    Science.gov (United States)

    Kremers, Gert-Jan; Piston, David

    2010-06-26

    Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles. Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion. This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

  5. Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

    Science.gov (United States)

    Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

    2013-11-01

    Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

  6. Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding

    Science.gov (United States)

    Hagelueken, Gregor; Naismith, James H

    2013-01-01

    Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins. PMID:24091556

  7. Targeting vascular amyloid in arterioles of Alzheimer disease transgenic mice with amyloid β protein antibody-coated nanoparticles.

    Science.gov (United States)

    Poduslo, Joseph F; Hultman, Kristi L; Curran, Geoffry L; Preboske, Gregory M; Chamberlain, Ryan; Marjańska, Małgorzata; Garwood, Michael; Jack, Clifford R; Wengenack, Thomas M

    2011-08-01

    The relevance of cerebral amyloid angiopathy (CAA) to the pathogenesis of Alzheimer disease (AD) and dementia in general emphasizes the importance of developing novel targeting approaches for detecting and treating cerebrovascular amyloid (CVA) deposits. We developed a nanoparticle-based technology that uses a monoclonal antibody against fibrillar human amyloid-β42 that is surface coated onto a functionalized phospholipid monolayer. We demonstrate that this conjugated nanoparticle binds to CVA deposits in arterioles of AD transgenic mice (Tg2576) after infusion into the external carotid artery using 3 different approaches. The first 2 approaches use a blood vessel enrichment of homogenized brain and a leptomeningeal vessel preparation from thin tangential brain slices from the surface of the cerebral cortex. Targeting of CVA by the antibody-coated nanoparticle was visualized using fluorescent lissamine rhodamine-labeled phospholipids in the nanoparticles, which were compared with fluorescent staining of the endothelial cells and amyloid deposits using confocal laser scanning microscopy. The third approach used high-field strength magnetic resonance imaging of antibody-coated iron oxide nanoparticles after infusion into the external carotid artery. Dark foci of contrast enhancement in cortical arterioles were observed in T2*-weighted images of ex vivo AD mouse brains that correlated histologically with CVA deposits. The targeting ability of these nanoparticles to CVA provides opportunities for the prevention and treatment of CAA.

  8. Ectopic bone formation cannot occur by hydroxyapatite/β-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    Science.gov (United States)

    Cheng, Lijia; Duan, Xin; Xiang, Zhou; Shi, Yujun; Lu, Xiaofeng; Ye, Feng; Bu, Hong

    2012-12-01

    Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/β-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede osteoinduction by CP. Overall, our results may shed light on clear mechanism study of bone induction in the future.

  9. Ectopic bone formation cannot occur by hydroxyapatite/{beta}-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    Energy Technology Data Exchange (ETDEWEB)

    Cheng Lijia [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Duan Xin [Department of Orthopaedics, Chengdu Second People' s Hospital, Chengdu (China); Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Xiang Zhou [Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Shi Yujun; Lu Xiaofeng; Ye Feng [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Bu Hong, E-mail: hongbu@scu.edu.cn [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Department of Pathology, West China Hospital, Sichuan University, Chengdu (China)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer Firstly, chimeric mouse model could be established successfully by bone marrow transplantation after irradiation. Black-Right-Pointing-Pointer Secondly, bone induction can occur in wild-type mice 90 days after implantation, but not occur in chimeric mice. Black-Right-Pointing-Pointer Thirdly, destruction of immune function will block osteoinduction by calcium phosphate ceramics. - Abstract: Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/{beta}-tricalcium phosphate (HA/{beta}-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/{beta}-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede

  10. ZnO nanoparticles assist the refolding of denatured green fluorescent protein.

    Science.gov (United States)

    Pandurangan, Muthuraman; Zamany, Ahmad Jawid; Kim, Doo Hwan

    2016-04-01

    Proteins are essential for cellular and biological processes. Proteins are synthesized and fold into the native structure to become active. The inability of a protein molecule to remain in its native conformation is called as protein misfolding, and this is due to several environmental factors. Protein misfolding and aggregation handle several human diseases. Protein misfolding is believed to be one of the causes of several disorders such as cancer, degenerative diseases, and metabolic pathologies. The zinc oxide (ZnO) nanoparticle was significantly promoted refolding of thermally denatured green fluorescent protein (GFP). In the present study, ZnO nanoparticles interaction with GFP was investigated by ultraviolet-visible spectrophotometer, fluorescence spectrophotometer, and dynamic light scattering. Results suggest that the ZnO nanoparticles significantly assist the refolding of denatured GFP. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Hepatitis B virus HBx protein impairs liver regeneration through enhanced expression of IL-6 in transgenic mice.

    Science.gov (United States)

    Quétier, Ivan; Brezillon, Nicolas; Duriez, Marion; Massinet, Hélène; Giang, Eric; Ahodantin, James; Lamant, Céline; Brunelle, Marie-Noëlle; Soussan, Patrick; Kremsdorf, Dina

    2013-08-01

    Conflicting results have been reported regarding the impact of hepatitis B virus X protein (HBx) expression on liver regeneration triggered by partial hepatectomy (PH). In the present report we investigated the mechanisms by which HBx protein alters hepatocyte proliferation after PH. PH was performed on a transgenic mouse model in which HBx expression is under the control of viral regulatory elements and liver regeneration was monitored. LPS, IL-6 neutralizing antibody, and SB203580 were injected after PH to evaluate IL-6 participation during liver regeneration. Cell cycle progression of hepatocytes was delayed in HBx transgenic mice compared to WT animals. Moreover, HBx induced higher secretion of IL-6 soon after PH. Upregulation of IL-6 was associated with an elevation of STAT3 phosphorylation, SOCS3 transcript accumulation and a decrease in ERK1/2 phosphorylation in the livers of HBx transgenic mice. The involvement of IL-6 overexpression in cell cycle deregulation was confirmed by the inhibition of liver regeneration in control mice after the upregulation of IL-6 expression using LPS. In addition, IL-6 neutralization with antibodies was able to restore liver regeneration in HBx mice. Finally, the direct role of p38 in IL-6 secretion after PH was demonstrated using SB203580, a pharmacological inhibitor. HBx is able to induce delayed hepatocyte proliferation after PH, and HBx-induced IL-6 overexpression is involved in delayed liver regeneration. By modulating IL-6 expression during liver proliferation induced by stimulation of the cellular microenvironment, HBx may participate in cell cycle deregulation and progression of liver disease. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  12. Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Valero Francisco

    2007-05-01

    Full Text Available Abstract Background Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a Rhizopus oryzae lipase (ROL in Pichia pastoris by means of 2D-fluorimetric techniques Results In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in P. pastoris batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity Conclusion P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in P. pastoris using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes.

  13. Expression Systems and Species Used for Transgenic Animal Bioreactors

    OpenAIRE

    Yanli Wang; Sihai Zhao; Liang Bai; Jianglin Fan; Enqi Liu

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm ...

  14. Expression Systems and Species Used for Transgenic Animal Bioreactors

    OpenAIRE

    Yanli Wang; Sihai Zhao; Liang Bai; Jianglin Fan; Enqi Liu

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm ...

  15. Rise-time of FRET-acceptor fluorescence tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Van Mierlo, C.P.M.; Visser, A.J.W.G.; Borst, J.W.

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no correspon

  16. Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

    Science.gov (United States)

    Ingersoll, Christine M.; Strollo, Christen M.

    2007-01-01

    The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

  17. Drug/protein interactions studied by time-resolved fluorescence spectroscopy

    Science.gov (United States)

    Gustavsson, Thomas; Markovitsi, Dimitra; Vayá, Ignacio; Bonancía, Paula; Jiménez, M. C.; Miranda, Miguel A.

    2014-09-01

    We report here on a recent time-resolved fluorescence study [1] of the interaction between flurbiprofen (FBP), a chiral non-steroidal anti-inflammatory drug, and human serum albumin (HSA), the main transport protein in the human body. We compare the results obtained for the drug-protein complex with those of various covalently linked flurbiprofentryptophan dyads having well-defined geometries. In all cases stereoselective dynamic fluorescence quenching is observed, varying greatly from one system to another. In addition, the fluorescence anisotropy decays also display a clear stereoselectivity. For the drug-protein complexes, this can be interpreted in terms of the protein microenvironment playing a significant role in the conformational relaxation of FBP, which is more restricted in the case of the (R)- enantiomer.

  18. Time-Resolved Fluorescence Immunoassay for C-Reactive Protein Using Colloidal Semiconducting Nanoparticles

    Directory of Open Access Journals (Sweden)

    Pekka Hänninen

    2011-11-01

    Full Text Available Besides the typical short-lived fluorescence with decay times in the nanosecond range, colloidal II/VI semiconductor nanoparticles dispersed in buffer also possess a long-lived fluorescence component with decay times in the microsecond range. Here, the signal intensity of the long-lived luminescence at microsecond range is shown to increase 1,000-fold for CdTe nanoparticles in PBS buffer. This long-lived fluorescence can be conveniently employed for time-gated fluorescence detection, which allows for improved signal-to-noise ratio and thus the use of low concentrations of nanoparticles. The detection principle is demonstrated with a time-resolved fluorescence immunoassay for the detection of C-reactive protein (CRP using CdSe-ZnS nanoparticles and green light excitation.

  19. Study on Spatial-temporal Dynamics of Bt Toxic Protein Expression in Insect-resistant Transgenic Cotton and Its Degradation in Soil

    Institute of Scientific and Technical Information of China (English)

    Yiwen ZHANG; Lianrong WANG; Liancheng ZHANG; Jun ZHANG; Xinbo JI; Jinmao WANG

    2012-01-01

    [Ob.jcctive] This study aimed to investigate the spatial-temporal dynamics of Bt toxic protein expression in insect-resistant transgenic cotton and its degradation in soil. [Meth~d] BtcrylAc toxic protein expression in roots, stems and leaves of trans- genic cotton Guoshen GK45 at different developmental stages and the annual aver- age content of BtCrylAc toxin protein in the topsoil, rhizosphere soil and following cotton-growing area were explored and analyzed by using enzyme linked immuno sorbed assay (ELISA). [Result] The content of exogenous BtCrylAc toxin protein de- creased during the growth process of insect-resistant transgenic cotton; to be specif- ic, the content of BtCrylAc toxin protein in cotton stems and leaves decreased more slowly and always maintained a high level, while that in roots decreased rapidly and reached a minimum level to the following plant growth and development stage. BtCrylAc toxin protein was detected in topsoil of both non-transgenic and transgenic cotton-growing areas, and the content of BtCrylAc toxin protein increased in topsoil of following cotton-growing area, which was very low in rhizosphere soil. [Conclusion] Determination of BtcrylAc toxic protein provides scientific basis for the risk assess- ment of the cultivation of genetically modified crops and the safety evaluation of soil ecosystem.

  20. Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color FRET microscopy

    Science.gov (United States)

    Sun, Yuansheng; Booker, Cynthia F.; Day, Richard N.; Periasamy, Ammasi

    2009-02-01

    Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However, the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy. Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor photobleaching FRET microscopy.

  1. Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

    Science.gov (United States)

    Griffiths, N; Jaipargas, E-A; Wozny, M R; Barton, K A; Mathur, N; Delfosse, K; Mathur, J

    2016-08-01

    Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed.

  2. A transgenic Drosophila model demonstrates that the Helicobacter pylori CagA protein functions as a eukaryotic Gab adaptor.

    Directory of Open Access Journals (Sweden)

    Crystal M Botham

    2008-05-01

    Full Text Available Infection with the human gastric pathogen Helicobacter pylori is associated with a spectrum of diseases including gastritis, peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. The cytotoxin-associated gene A (CagA protein of H. pylori, which is translocated into host cells via a type IV secretion system, is a major risk factor for disease development. Experiments in gastric tissue culture cells have shown that once translocated, CagA activates the phosphatase SHP-2, which is a component of receptor tyrosine kinase (RTK pathways whose over-activation is associated with cancer formation. Based on CagA's ability to activate SHP-2, it has been proposed that CagA functions as a prokaryotic mimic of the eukaryotic Grb2-associated binder (Gab adaptor protein, which normally activates SHP-2. We have developed a transgenic Drosophila model to test this hypothesis by investigating whether CagA can function in a well-characterized Gab-dependent process: the specification of photoreceptors cells in the Drosophila eye. We demonstrate that CagA expression is sufficient to rescue photoreceptor development in the absence of the Drosophila Gab homologue, Daughter of Sevenless (DOS. Furthermore, CagA's ability to promote photoreceptor development requires the SHP-2 phosphatase Corkscrew (CSW. These results provide the first demonstration that CagA functions as a Gab protein within the tissue of an organism and provide insight into CagA's oncogenic potential. Since many translocated bacterial proteins target highly conserved eukaryotic cellular processes, such as the RTK signaling pathway, the transgenic Drosophila model should be of general use for testing the in vivo function of bacterial effector proteins and for identifying the host genes through which they function.

  3. Bodipy-FL-Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins

    Directory of Open Access Journals (Sweden)

    Anna Rosati

    2004-01-01

    Full Text Available Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV, have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR or MRP (multidrug resistance‐related protein (PANC‐1 and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine or MRP (MK571 and probenecid has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

  4. Fluorescence Lifetime Imaging of Free and Protein-Bound NADH

    Science.gov (United States)

    Lakowicz, Joseph R.; Szmacinski, Henryk; Nowaczyk, Kazimierz; Johnson, Michael L.

    1992-02-01

    We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challenging case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images, each obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe an imaging procedure that allows specific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.

  5. A green fluorescent protein with photoswitchable emission from the deep sea.

    Directory of Open Access Journals (Sweden)

    Alexander Vogt

    Full Text Available A colorful variety of fluorescent proteins (FPs from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

  6. Mass Spectrometric Imaging of Red Fluorescent Protein in Breast Tumor Xenografts

    Science.gov (United States)

    Chughtai, Kamila; Jiang, Lu; Post, Harm; Winnard, Paul T.; Greenwood, Tiffany R.; Raman, Venu; Bhujwalla, Zaver M.; Heeren, Ron M. A.; Glunde, Kristine

    2013-05-01

    Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters.

  7. Environmental and transgene expression effects on the barley seed proteome

    DEFF Research Database (Denmark)

    Finnie, Christine; Steenholdt, T.; Noguera, O.R.;

    2004-01-01

    The barley (Hordeum vulgare) cultivar Golden Promise is no longer widely used for malting, but is amenable to transformation and is therefore a valuable experimental cultivar. Its characteristics include high salt tolerance, however it is also susceptible to several fungal pathogens. Proteome....... Eleven of these were identified by mass spectrometric peptide mass mapping, including an abundant chitinase implicated in defence against fungal pathogens and a small heat-shock protein. To enable a comparison with transgenic seed protein patterns, differences in spot patterns between field...... with extra nitrogen. Finally, the fate of transgene products in barley seeds was followed. Spots containing two green fluorescent protein constructs and the herbicide resistance marker phosphinothricin acetyltransferase were observed in 2D-gel patterns of transgenic seeds and identified by mass spectrometry...

  8. Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

    Science.gov (United States)

    Köhler, Stephan; Ouahrani-Bettache, Safia; Layssac, Marion; Teyssier, Jacques; Liautard, Jean-Pierre

    1999-01-01

    A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA to gfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized. PMID:10569794

  9. Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

    OpenAIRE

    Köhler, Stephan; Ouahrani-Bettache, Safia; Layssac, Marion; Teyssier, Jacques; Liautard, Jean-Pierre

    1999-01-01

    A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA to gfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

  10. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Jae Eun Lee

    2015-06-01

    Full Text Available Two dimensional-fluorescence difference gel electrophoresis (2D DIGE is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

  11. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Science.gov (United States)

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

  12. Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching.

    Science.gov (United States)

    Iijima, Issei; Hohsaka, Takahiro

    2009-04-17

    Position-specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. We have developed a method for the incorporation of visible-wavelength-fluorescent non-natural amino acids into proteins in a cell-free translation system. Using this technique, we introduced one or two BODIPY-linked amino acids into maltose-binding protein (MBP) to obtain MBP derivatives showing ligand-dependent changes in fluorescence intensity or intensity ratio. BODIPY-FL-aminophenylalanine was incorporated in place of 15 tyrosines, as well as the N-terminal Lys1, and the C-terminal Lys370 of MBP. Fluorescence measurements revealed that MBP containing a BODIPY-FL moiety in place of Tyr210 showed a 13-fold increase in