Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.
- Klíčová slova
- Confocal microscopy, Embryo, Green fluorescent protein (GFP), Heart, Optical projection tomography, Tissue transparency,
- MeSH
- myši transgenní MeSH
- myši MeSH
- srdce embryologie MeSH
- zelené fluorescenční proteiny analýza biosyntéza genetika MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- zelené fluorescenční proteiny MeSH
GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.
- MeSH
- benzylové sloučeniny chemie MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA-dependentní DNA-polymerasy chemie metabolismus MeSH
- DNA chemie metabolismus MeSH
- fluorescenční barviva chemie MeSH
- imidazoliny MeSH
- imidazoly chemie MeSH
- molekulární sekvence - údaje MeSH
- nukleotidy chemie metabolismus MeSH
- párování bází MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone MeSH Prohlížeč
- 3,5-difluoro-4-hydroxybenzylidene imidazolinone MeSH Prohlížeč
- benzylové sloučeniny MeSH
- DNA vazebné proteiny MeSH
- DNA-dependentní DNA-polymerasy MeSH
- DNA MeSH
- fluorescenční barviva MeSH
- imidazoliny MeSH
- imidazoly MeSH
- nukleotidy MeSH
- Tli polymerase MeSH Prohlížeč
Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.
- MeSH
- biologický transport MeSH
- exprese genu MeSH
- HEK293 buňky MeSH
- klonování DNA MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací MeSH
- nanočástice chemie metabolismus MeSH
- penetrační peptidy metabolismus MeSH
- plazmidy genetika MeSH
- průtoková cytometrie MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus ultrastruktura MeSH
- transfekce MeSH
- western blotting MeSH
- zelené fluorescenční proteiny genetika izolace a purifikace metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- penetrační peptidy MeSH
- rekombinantní proteiny MeSH
- zelené fluorescenční proteiny MeSH
An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns.
- Klíčová slova
- Flow cytometry, Fluorescence intensity, Green fluorescent protein, Inducible promoter, Nitrate, Nitrogen source,
- Publikační typ
- časopisecké články MeSH
Green Fluorescent protein (GFP) transgenic animals are accepted tools for studying various physiological processes, including organ development and cell migration. However, several in vivo studies claimed that GFP may impair transgenic animals' health. Glomerulosclerosis was observed in transgenic mice and rabbits with ubiquitous reporter protein expression. Heart-specific GFP expression evoked dilated cardiomyopathy and altered cardiac function in transgenic mouse and zebrafish lines, respectively. Moreover, growth retardation and increased axon swelling were observed in GFP and yellow fluorescent protein (YFP) transgenic mice, respectively. This review will focus on the potential drawbacks of the applications of GFP transgenic animals in biomedical research.
- MeSH
- biomedicínský výzkum metody trendy MeSH
- dánio pruhované MeSH
- geneticky modifikovaná zvířata genetika MeSH
- králíci MeSH
- myši transgenní MeSH
- myši MeSH
- vývojová regulace genové exprese MeSH
- zelené fluorescenční proteiny biosyntéza genetika MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- zelené fluorescenční proteiny MeSH
Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment-protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound 'free' proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 - 2.95 µm2s-1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50-500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII-light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein-protein interactions in the mobility restrictions for large thylakoid protein complexes.
- Klíčová slova
- FCS, cyanobacteria, photosynthesis, proteins mobility, thylakoids,
- Publikační typ
- časopisecké články MeSH
The development of the root apex is determined by progress of cells from the meristematic region to the successive post-mitotic developmental zones for transition, cell elongation and final cell differentiation. We addressed root development, tissue architecture and root developmental zonation by means of light-sheet microscopic imaging of Arabidopsis thaliana seedlings expressing END BINDING protein 1c (EB1c) fused to green fluorescent protein (GFP) under control of native EB1c promoter. Unlike the other two members of the EB1 family, plant-specific EB1c shows prominent nuclear localization in non-dividing cells in all developmental zones of the root apex. The nuclear localization of EB1c was previously mentioned solely in meristematic cells, but not further addressed. With the help of advanced light-sheet microscopy, we report quantitative evaluations of developmentally-regulated nuclear levels of the EB1c protein tagged with GFP relatively to the nuclear size in diverse root tissues (epidermis, cortex, and endodermis) and root developmental zones (meristem, transition, and elongation zones). Our results demonstrate a high potential of light-sheet microscopy for 4D live imaging of fluorescently-labeled nuclei in complex samples such as developing roots, showing capacity to quantify parameters at deeper cell layers (e.g., endodermis) with minimal aberrations. The data presented herein further signify the unique role of developmental cell reprogramming in the transition from cell proliferation to cell differentiation in developing root apex.
- Klíčová slova
- development, end-binding 1c (EB1c), light-sheet microscopy, nucleus, root apex, transition zone,
- Publikační typ
- časopisecké články MeSH
Azaarenes are nitrogen-containing polyaromatic heterocyclic compounds (NPAHs). The majority of the azaarenes found in the environment originate from anthropogenic sources. Concentrations of NPAHs found in the environment are reported to be one to two orders of magnitude lower than polycyclic aromatic hydrocarbons (PAHs) concentrations, yet their biological effects can be of similar magnitude. Very few studies on the genotoxicity of azaarenes are available in the literature. In the present study, a preliminary profile of both the toxic and genotoxic potential of 5 PAHs and their 20 aza-analogues were investigated. To assess the toxic and genotoxic activity, a green fluorescent protein (GFP) assay based on the yeast Saccharomyces cerevisiae was selected. To compare the sensitivity of this eukaryotic short-term assay with bacterial screening tests, the Toxi-Chromotest for toxicity and SOS-Chromotest for genotoxicity assessment were also performed. This comparison indicates that in most cases, the yeast GFP assay is apparently of comparable specificity to the bacterial toxicity or genotoxicity tests with respect to the correlation of positive/negative responses, but much more sensitive with respect to the effective concentration values. In the cases of phenazine, phenanthridine, 1,10-phenanthroline, or 4,7-phenanthroline, one to two orders of magnitude lower IC20 and minimum genotoxic concentration values in the yeast GFP assay were observed. In this study, the authors present evidence that genotoxicity assessment using the yeast GFP assay can provide a simple system to monitor the activity of these environmental pollutants that could possess mutagenic potential at low concentrations.
- MeSH
- biotest metody MeSH
- heterocyklické sloučeniny chemie toxicita MeSH
- látky znečišťující životní prostředí chemie toxicita MeSH
- monitorování životního prostředí metody MeSH
- Saccharomyces genetika MeSH
- testy genotoxicity metody MeSH
- zelené fluorescenční proteiny analýza MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- heterocyklické sloučeniny MeSH
- látky znečišťující životní prostředí MeSH
- zelené fluorescenční proteiny MeSH
Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.
- MeSH
- biologický transport MeSH
- buněčná membrána metabolismus MeSH
- fluorescenční barviva metabolismus MeSH
- genetické vektory genetika MeSH
- genové produkty gag genetika metabolismus MeSH
- HEK293 buňky MeSH
- kapsida metabolismus MeSH
- kinetika MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus genetika metabolismus fyziologie MeSH
- mikrotubuly metabolismus virologie MeSH
- molekulární zobrazování MeSH
- pohyb MeSH
- proviry genetika metabolismus fyziologie MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- sestavení viru MeSH
- transport proteinů MeSH
- viabilita buněk MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- fluorescenční barviva MeSH
- genové produkty gag MeSH
- rekombinantní fúzní proteiny MeSH
- zelené fluorescenční proteiny MeSH
Circulating tumor cells (CTCs) are potential precursors of metastasis. They are also of use in diagnosing malignancy and for prognostic purposes. Our laboratory has previously isolated CTCs from orthotopic nude mouse models of human prostate cancer cells where the PC-3 cancer cells express green fluorescent protein (GFP). It was found that orthotopic tumors produced CTCs and not subcutaneous tumors, which may explain why orthotopic tumors metastasize and subcutaneous tumors do not. However, in this previous study, CTCs were observed only after culture. In the present study, using the GFP-expressing PC-3 orthotopic model and immunomagnetic beads coated with anti-epithelial cell adhesion molecule (EpCAM) and anti-prostate specific membrane antigen (PSMA), GFP-expressing CTC were isolated within 15 minutes and were readily visualized by GFP fluorescence. It was possible to immediately place the immunomagnetic-bead-captured GFP-expressing PC-3 CTCs in 3-dimensional sponge cell culture, where they proliferated. The combination of GFP expression and the use of immunomagnetic beads is a very powerful method to obtain CTCs for either immediate analysis or for biological characterization in vivo or in 3-dimensional culture.
- MeSH
- adhezní molekula epiteliálních buněk MeSH
- antigeny nádorové imunologie MeSH
- glutamátkarboxypeptidasa II imunologie MeSH
- imunomagnetická separace * MeSH
- lidé MeSH
- lymfatické metastázy MeSH
- molekuly buněčné adheze imunologie MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- nádorové cirkulující buňky patologie MeSH
- nádory prostaty metabolismus patologie MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adhezní molekula epiteliálních buněk MeSH
- antigeny nádorové MeSH
- glutamátkarboxypeptidasa II MeSH
- molekuly buněčné adheze MeSH
- zelené fluorescenční proteiny MeSH
