GFP-expression
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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
- 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
Many studies have demonstrated the importance of spontaneous metastases in cancer research. Until now, we still had only a few spontaneous metastatic models with high occurrence rate of metastasis in distant lymph and visceral tissues. We report a syngeneic heterotopic metastatic model using the Lewis lung cancer cell line with high metastatic ratio in C57BL/6 mice after transplantation by injection of cancer cells and without surgical intervention. Metastatic process was declared for each mouse in two groups ?sacrificed 3 or 5 weeks after subcutaneous (s.c.) injection of the tumor cells into the dorsal side of the tail. The total number of metastases was counted as the sum of observed macrometastases. Our model produced produced a 100% rate of spontaneous lymphatic and visceral metastases after a simple injection transplantation into the heterotopic site. In mice with large primary tumors which are non-lethal, visceral and lymph macrometastases were observed. Tumor volume correlated linearly not only with the tumor growth time, but also with the number of metastases in lymph nodes and organs. This new metastatic model could be useful for studying the metastasis mechanism and for developing therapy for lymph and visceral metastases.
Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.
- MeSH
- glykosylace MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- subcelulární frakce MeSH
- tetraspaniny genetika metabolismus MeSH
- transport proteinů MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Genetické modifikace (GM) hmyzu zahrnují řadu metod vedoucích k odstranění (umlčení) určitého genu (nejčastěji pomocí RNAi, nebo knockouť genu) nebo vnesení cizorodého genu (hlavně pomocí transpozonů, z nichž je nejznámější systém P-elementů). V obou případech se úspěšnost GM sleduje pomocí exprese „marker" genů (dnes většinou „green fluorescent protein"). Pro drozofilu jsou dostupné další techniky a zejména velké banky různých mutací. Systém cis elementu UAS („Upstream Activation Sequence") a jeho aktivátoru Gal4 umožňuje exprimovat vnesený gen v určitých buňkách či orgánech. Genetické modifikace u drozofily i dalších modelových druhů hmyzu se používají jako prostředek pro objasnění vývojových a fyziologických regulací, včetně poruch, které napodobují lidská onemocnění. GM mohou také omezit rozmnožování, nebo způsobit úhyn škodlivého hmyzu. Tato technika by však vyžadovala vypouštění GM hmyzu do přírody a to je v současnosti nemožné. Prakticky významné jsou však GM využívané v chovech hmyzu, který je po sterilizaci vypouštěn do přírody, aby svým přečíslením podstatně snížil rozmnožování přírodní populace (tzv. „sterile insect technice", SIT).
Genetic modifications of insects include diverse methods of gene silencing, such as gene knockdown or RNAi, and methods of transgenesis when a foreign gene is introduced into insect genome, usually with the aid of transponsons such as the P elements. The success of GM is typically visualized through expression of a gene encoding the green fluorescent protein or another marker. Additional techniques and also large banks of diverse mutations are available for drosophila. The system of UAS (Upstream Activation Sequence) and its transcription activator Gal4 allows in this species transgene expression in certain cells and organs. Genetic modifications of drosophila and other model insect species provide efficient tool for elucidating developmental and physiological regulations, including defects mimicking human diseases. GM can also be used to curb insect reproduction or survival. Practical deployment of this approach, however, would require release of GM insects into the environment and this would hardly be permitted. On the other hand, GMs restricted to insect cultures are practically exploited in the sterile insect technique (SIT), when sterilized, non-GM insects are released into the field and due to their high numbers and random mating suppress the growth of the native wild population.
- Klíčová slova
- umlčení genů, transgeneze, P-elementy, Piggy bag, GFP, UAS/Gal4, vypouštění sterilních samců,
- MeSH
- financování organizované MeSH
- geneticky modifikovaná zvířata genetika MeSH
- hmyz genetika MeSH
- regulace genové exprese genetika MeSH
- RNA interference MeSH
- technika přenosu genů MeSH
- transgeny fyziologie genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata 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
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.
- 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
To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP-Cerulean/DMP1-Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24-258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1-Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP-Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.
- MeSH
- buněčná diferenciace MeSH
- extracelulární matrix - proteiny genetika MeSH
- fluorescenční barviva MeSH
- fosfoproteiny genetika MeSH
- myši transgenní MeSH
- myši MeSH
- odontoblasty cytologie MeSH
- reportérové geny * MeSH
- sialoglykoproteiny genetika MeSH
- transgeny MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
The mouse Dach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles (LV) of the adult mouse brain. In this study we aimed to elucidate the role of Dach1-expressing cells in adult neurogenesis/gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mouse Dach1 gene. A neurosphere-forming assay of GFP⁺ cells isolated from the dorsal part of the LV was carried out with subsequent differentiation in vitro. To elucidate the neurogenic/gliogenic potential of GFP⁺ cells in the dorsal part of the LV, in situ immunohistochemical/electrophysiological analyses of GFP⁺ cells in adult sham-operated brains (controls) and those after middle cerebral artery occlusion (MCAo) were performed. The GFP⁺ cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated solely into a glial phenotype, while those isolated after MCAo also gave rise to cells with the properties of neuronal precursors. In situ analyses revealed that GFP⁺ cells express the phenotype of adult NSCs or neuroblasts in controls as well as following ischemia. Following MCAo we found a significantly increased number of GFP⁺ cells expressing doublecortin as well as a number of GFP⁺ cells migrating through the rostral migratory stream into the olfactory bulb, where they probably differentiated into calretinin⁺ interneurons. Collectively, our results suggest the involvement of the mouse Dach1 gene in adult neurogenesis; cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.
- MeSH
- 4-aminopyridin farmakologie MeSH
- blokátory sodíkových kanálů farmakologie MeSH
- buněčná diferenciace fyziologie MeSH
- degenerace nervu etiologie patologie MeSH
- dospělé kmenové buňky fyziologie MeSH
- infarkt arteria cerebri media komplikace MeSH
- membránové potenciály účinky léků MeSH
- metoda terčíkového zámku MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- myši MeSH
- neurogeneze fyziologie MeSH
- neurony metabolismus MeSH
- oční proteiny metabolismus MeSH
- počet buněk MeSH
- proteiny nervové tkáně metabolismus MeSH
- techniky in vitro MeSH
- tetraethylamonium farmakologie MeSH
- tetrodotoxin farmakologie MeSH
- ventriculi laterales patologie MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901-3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato-GFP proteins associate with detergent-resistant membranes; two of them, Ato1p-GFP and Ato3p-GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p-GFP which forms stable patches, the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p-GFP clustering and patches spreading are reversible. The Ato1p-GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.
- MeSH
- amoniak metabolismus MeSH
- buněčná membrána metabolismus MeSH
- detergenty metabolismus MeSH
- financování organizované MeSH
- kompartmentace buňky MeSH
- koncentrace vodíkových iontů MeSH
- kvartérní amoniové sloučeniny metabolismus MeSH
- membránové proteiny metabolismus MeSH
- membránové transportní proteiny metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae cytologie metabolismus MeSH
- transport proteinů MeSH
- zelené fluorescenční proteiny metabolismus MeSH
