Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent protein...Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent proteins targeted to specific subcellular structures to probe localmolecular environment,which opens new directions in cell science.This paper highlights theunconventional applications of FLIM for studies of molecular processes in diverse organelles oflive cultured cells.展开更多
A new reversibly switchable fluorescent protein(RSFP), namely Dreiklang, exhibits prominent feature that the wavelengths for switching and fluorescence are decoupled due to its great different structures between bri...A new reversibly switchable fluorescent protein(RSFP), namely Dreiklang, exhibits prominent feature that the wavelengths for switching and fluorescence are decoupled due to its great different structures between bright and dark states. This feature might also induce some nonlinear optic(NLO) properties changing as switching between two states, which might promote new method of biological science. We employ the QM/MM method to simulate the structures of different states, and study their second harmonic generation(SHG) and two-photon absorption(TPA) properties. And we found different states of Dreiklang have different SHG and TPA responses. The SHG and TPA properties of Dreiklang are correlated to particularly geometrical structures of different states, especially the centrosymmetric or nocentrosymmetric π-stacking structures which are formed by chromophore and beside residue Tyr203, so the SHG and TPA responses could be changed as the light induces switching among different states of Dreiklang. This work would prospectively guide the application of Dreiklang on the NLO technology, and help the development of new RSFP with special NLO function.展开更多
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 intere...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 dy- namic 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.展开更多
A feasible method of combining the concept of fluorescence half-life and the power dependent photo- bleaching rate for characterizing the practical photostability of fluorescent proteins (FPs) was introduced. Furthe...A feasible method of combining the concept of fluorescence half-life and the power dependent photo- bleaching rate for characterizing the practical photostability of fluorescent proteins (FPs) was introduced. Furthermore, by using a fluorescent photostability standard, a relative comparison of the photostabilty of FPs from different research groups was proposed, which would be of great benefit for developing novel FPs with optimized emission wavelength, better brightness, and improved photostability. We used rho- damine B as an example to verify this method and evaluate the practical photostability of a far-red FP, mKate-S158C. Experimental results indicated good potential of this method for further study.展开更多
Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and supe...Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the 'positively switchable' PADRON C-s with the 'negatively switchable' rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.展开更多
Stroke can cause Wallerian degeneration in regions outside of the brain,particularly in the corticospinal tract.To investigate the fate of major glial cells and axons within affected areas of the corticospinal tract f...Stroke can cause Wallerian degeneration in regions outside of the brain,particularly in the corticospinal tract.To investigate the fate of major glial cells and axons within affected areas of the corticospinal tract following stroke,we induced photochemical infarction of the sensorimotor cortex leading to Wallerian degeneration along the full extent of the corticospinal tract.We first used a routine,sensitive marker of axonal injury,amyloid precursor protein,to examine Wallerian degeneration of the corticospinal tract.An antibody to amyloid precursor protein mapped exclusively to proximal axonal segments within the ischemic cortex,with no positive signal in distal parts of the corticospinal tract,at all time points.To improve visualization of Wallerian degeneration,we next utilized an orthograde virus that expresses green fluorescent protein to label the corticospinal tract and then quantitatively evaluated green fluorescent protein-expressing axons.Using this approach,we found that axonal degeneration began on day 3 post-stroke and was almost complete by 7 days after stroke.In addition,microglia mobilized and activated early,from day 7 after stroke,but did not maintain a phagocytic state over time.Meanwhile,astrocytes showed relatively delayed mobilization and a moderate response to Wallerian degeneration.Moreover,no anterograde degeneration of spinal anterior horn cells was observed in response to Wallerian degeneration of the corticospinal tract.In conclusion,our data provide evidence for dynamic,pathogenic spatiotemporal changes in major cellular components of the corticospinal tract during Wallerian degeneration.展开更多
Aim The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells (DPSCs) from green fluorescent protein (GFP) transgenic mice. The expression of GFP in DPSCs was also obs...Aim The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells (DPSCs) from green fluorescent protein (GFP) transgenic mice. The expression of GFP in DPSCs was also observed during differentiation. Methodology DPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR. Results The endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation. Conclusion As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.展开更多
Brain-derived neurotrophic factor(BDNF)can significantly promote nerve regeneration and repair.High expression of the BDNF-green fluorescent protein(GFP)gene persists for a long time after transfection into neural...Brain-derived neurotrophic factor(BDNF)can significantly promote nerve regeneration and repair.High expression of the BDNF-green fluorescent protein(GFP)gene persists for a long time after transfection into neural stem cells.Nevertheless,little is known about the biological characteristics of BDNF-GFP modified nerve stem cells in vivo and their ability to induce BDNF expression or repair spinal cord injury.In the present study,we transplanted BDNF-GFP transgenic neural stem cells into a hemisection model of rats.Rats with BDNF-GFP stem cells exhibited significantly increased BDNF expression and better locomotor function compared with stem cells alone.Cellular therapy with BDNF-GFP transgenic stem cells can improve outcomes better than stem cells alone and may have therapeutic potential for spinal cord injury.展开更多
BACKGROUND: Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein (EGFP) in in vitro cultured retinal gangli...BACKGROUND: Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein (EGFP) in in vitro cultured retinal ganglial cells (RGCs). OBJECTIVE: To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING: In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS: Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS: A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls (n = 5) were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group (n = 15) was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group (n = 15), the eyes were irradiated with low-energy ultrasound wave (0.5 W/cm^2) for a total of 60 seconds (irradiated for 5 seconds, at 10-second intervals) immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group (n = 15), the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES: After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS: The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION: Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.展开更多
Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain...Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma.展开更多
Objective: To construct green fluorescent protein(GFP) retroviral vector(pLgXSN), and to investigate the expression of GFP in primary rat myoblast. Methods: GFP cDNA was subcloned into the plasmid pLgXSN, and th...Objective: To construct green fluorescent protein(GFP) retroviral vector(pLgXSN), and to investigate the expression of GFP in primary rat myoblast. Methods: GFP cDNA was subcloned into the plasmid pLgXSN, and the recombinant vector was transfected into packaging cell PT67. G418 was used to select positive colony. Myoblasts were infected by a high-titer viral supernatant. The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis. Confocal microscopy and flow cytometry were used to detect the expression of GFP. Results: The GFP cDNA sequence was identical to that of GenBank. Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully. The titer of the packaged recombinant retrovirus was 1×10^6 cfu/ml. Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection. The transfection rate was 33%. The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks. Conclusion: GFP gene could be effectively and stably expressed in myoblast, which suggests that GFP could act as a marker for studies on myoblast.展开更多
The human immunodeficiency virus (HIV) lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentivir...The human immunodeficiency virus (HIV) lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentiviral vector containing green fluorescent protein and vesicular stomatitis virus G pseudo-capsule was constructed. The plasmids were pHR-CMV-EGFP, pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G. The four plasmid system was co-transfected into 293T cells, and green fluorescent protein expression was observed. The present study obtained lentiviral particles by high-speed centrifugation, and the lentiviral particle titer was 4 × 108 TU/mL after centrifugation. Thus, an optimized novel HIV-1 lentiviral vector was successfully constructed.展开更多
We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constr...We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.展开更多
The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells...The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.展开更多
Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tum...Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.展开更多
The plasmid pGPDGFP under the control ofpgpdA promotor was used together with vector pAN7-1 containing the hygromycin resistance cassette to co-transform protoplasts of HG1, Fusarium graminearum from Hubei Province, C...The plasmid pGPDGFP under the control ofpgpdA promotor was used together with vector pAN7-1 containing the hygromycin resistance cassette to co-transform protoplasts of HG1, Fusarium graminearum from Hubei Province, China. Twelve out of 14 hygromycin-resistant transformants showed green signal under the UV light and contained one or several copies ofgfp, as indicated by Southern analysis of genomic DNA digested with different restriction enzymes and hybridized to the gfp probe. A single gfp copy transformant (HG1C5) was selected for further evaluation of 80 Chinese wheat cultivars or advanced lines. The results showed different resistance type to F. graminearum were observed. GFP signals observed in the rachis and adjacent spikes of 70 Chinese wheat lines such as Chuanchongzu 104 indicated both type I (host resistance to the initial infection by the fungus) and type II (resistance to the spread of FHB symptoms within an infected spike) were not observed. While other 10 lines showed type II resistance to F. graminearum with GFP signals only in inoculated spikelets. Development of the mycelium can be intuitively observed and the resistance of wheat to F. graminearum can be identified at 7 days post inoculation (dpi) in this way. The results showed no differences were evaluated between the transformed HG1C5 and the non-transgene artificial inoculation by SAS paired chi-square test and McNernar's test (P=-0.0625).展开更多
The green fluorescent protein (GFP) from jellyfishAequorea victoria has unique superiority as a kind of biological label. GFP has been widely used in all fields of biology based on the recent studies on its structure,...The green fluorescent protein (GFP) from jellyfishAequorea victoria has unique superiority as a kind of biological label. GFP has been widely used in all fields of biology based on the recent studies on its structure, characteristic and mechanism of bioluninescence.展开更多
In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected...In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.展开更多
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to invest...The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro , fluorescent VLPs could bind to CV 1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.展开更多
基金supported by the National Basic Research Program of China(2015CB352005)the National Natural Science Foundation of China(61525503/61378091/61620106016)+2 种基金Guangdong Natural Science Foundation Innovation Team(2014A030312008)Hong Kong,Macao and Taiwan cooperation innovation platform and major projects of international cooperation in Colleges and Universities in Guangdong Province(2015KGJHZ002)Shenzhen Basic Research Project(JCYJ20150930104948169/JCYJ20160328144746940/GJHZ 20160226202139185).
文摘Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent proteins targeted to specific subcellular structures to probe localmolecular environment,which opens new directions in cell science.This paper highlights theunconventional applications of FLIM for studies of molecular processes in diverse organelles oflive cultured cells.
基金based on work supported by the National Natural Science Foundation of China(No.21703246 and 21403242)Natural Science Foundation of Fujian Province(2014J05021)
文摘A new reversibly switchable fluorescent protein(RSFP), namely Dreiklang, exhibits prominent feature that the wavelengths for switching and fluorescence are decoupled due to its great different structures between bright and dark states. This feature might also induce some nonlinear optic(NLO) properties changing as switching between two states, which might promote new method of biological science. We employ the QM/MM method to simulate the structures of different states, and study their second harmonic generation(SHG) and two-photon absorption(TPA) properties. And we found different states of Dreiklang have different SHG and TPA responses. The SHG and TPA properties of Dreiklang are correlated to particularly geometrical structures of different states, especially the centrosymmetric or nocentrosymmetric π-stacking structures which are formed by chromophore and beside residue Tyr203, so the SHG and TPA responses could be changed as the light induces switching among different states of Dreiklang. This work would prospectively guide the application of Dreiklang on the NLO technology, and help the development of new RSFP with special NLO function.
文摘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 dy- namic 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.
基金supported by the National HighTech Research and Development Program of China(No.2006AA020801)the National Natural Science Foundation of China(No.30770525)the Programme of Introducing Talents of Discipline to Universities
文摘A feasible method of combining the concept of fluorescence half-life and the power dependent photo- bleaching rate for characterizing the practical photostability of fluorescent proteins (FPs) was introduced. Furthermore, by using a fluorescent photostability standard, a relative comparison of the photostabilty of FPs from different research groups was proposed, which would be of great benefit for developing novel FPs with optimized emission wavelength, better brightness, and improved photostability. We used rho- damine B as an example to verify this method and evaluate the practical photostability of a far-red FP, mKate-S158C. Experimental results indicated good potential of this method for further study.
文摘Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the 'positively switchable' PADRON C-s with the 'negatively switchable' rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.
基金supported by the National Natural Science Foundation of China,Nos.31 730030 (to XL),81941011 (to XL),31 771053 (to HD),82271403 (to XL),82272171 (to ZY),31971279 (to ZY)82201542 (to FH)+1 种基金the Natural Science Foundation of Beijing,No.7222004 (to HD)the Science and Technology Program of Beijing,No.Z181100001818007(to ZY)
文摘Stroke can cause Wallerian degeneration in regions outside of the brain,particularly in the corticospinal tract.To investigate the fate of major glial cells and axons within affected areas of the corticospinal tract following stroke,we induced photochemical infarction of the sensorimotor cortex leading to Wallerian degeneration along the full extent of the corticospinal tract.We first used a routine,sensitive marker of axonal injury,amyloid precursor protein,to examine Wallerian degeneration of the corticospinal tract.An antibody to amyloid precursor protein mapped exclusively to proximal axonal segments within the ischemic cortex,with no positive signal in distal parts of the corticospinal tract,at all time points.To improve visualization of Wallerian degeneration,we next utilized an orthograde virus that expresses green fluorescent protein to label the corticospinal tract and then quantitatively evaluated green fluorescent protein-expressing axons.Using this approach,we found that axonal degeneration began on day 3 post-stroke and was almost complete by 7 days after stroke.In addition,microglia mobilized and activated early,from day 7 after stroke,but did not maintain a phagocytic state over time.Meanwhile,astrocytes showed relatively delayed mobilization and a moderate response to Wallerian degeneration.Moreover,no anterograde degeneration of spinal anterior horn cells was observed in response to Wallerian degeneration of the corticospinal tract.In conclusion,our data provide evidence for dynamic,pathogenic spatiotemporal changes in major cellular components of the corticospinal tract during Wallerian degeneration.
基金funded by The Peabody Foundation Inc.,the Anthony and Constance Franchi Fund for Pediatric Orthopaedics at the Mass General Hospital for Children, and the National Natural Science Foundation of China (30801304)Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD 200977)
文摘Aim The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells (DPSCs) from green fluorescent protein (GFP) transgenic mice. The expression of GFP in DPSCs was also observed during differentiation. Methodology DPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR. Results The endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation. Conclusion As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.
基金the Natural Science Foundation of Liaoning Province, No. 20052096
文摘Brain-derived neurotrophic factor(BDNF)can significantly promote nerve regeneration and repair.High expression of the BDNF-green fluorescent protein(GFP)gene persists for a long time after transfection into neural stem cells.Nevertheless,little is known about the biological characteristics of BDNF-GFP modified nerve stem cells in vivo and their ability to induce BDNF expression or repair spinal cord injury.In the present study,we transplanted BDNF-GFP transgenic neural stem cells into a hemisection model of rats.Rats with BDNF-GFP stem cells exhibited significantly increased BDNF expression and better locomotor function compared with stem cells alone.Cellular therapy with BDNF-GFP transgenic stem cells can improve outcomes better than stem cells alone and may have therapeutic potential for spinal cord injury.
基金the National Natural Science Foundation of China,No.30430230
文摘BACKGROUND: Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein (EGFP) in in vitro cultured retinal ganglial cells (RGCs). OBJECTIVE: To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING: In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS: Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS: A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls (n = 5) were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group (n = 15) was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group (n = 15), the eyes were irradiated with low-energy ultrasound wave (0.5 W/cm^2) for a total of 60 seconds (irradiated for 5 seconds, at 10-second intervals) immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group (n = 15), the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES: After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS: The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION: Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.
基金This work was supported by a grant from the National Natural science foundation of China (No. 39970752).
文摘Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma.
文摘Objective: To construct green fluorescent protein(GFP) retroviral vector(pLgXSN), and to investigate the expression of GFP in primary rat myoblast. Methods: GFP cDNA was subcloned into the plasmid pLgXSN, and the recombinant vector was transfected into packaging cell PT67. G418 was used to select positive colony. Myoblasts were infected by a high-titer viral supernatant. The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis. Confocal microscopy and flow cytometry were used to detect the expression of GFP. Results: The GFP cDNA sequence was identical to that of GenBank. Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully. The titer of the packaged recombinant retrovirus was 1×10^6 cfu/ml. Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection. The transfection rate was 33%. The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks. Conclusion: GFP gene could be effectively and stably expressed in myoblast, which suggests that GFP could act as a marker for studies on myoblast.
基金the National Natural Science Foundation of China, No. 30770755
文摘The human immunodeficiency virus (HIV) lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentiviral vector containing green fluorescent protein and vesicular stomatitis virus G pseudo-capsule was constructed. The plasmids were pHR-CMV-EGFP, pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G. The four plasmid system was co-transfected into 293T cells, and green fluorescent protein expression was observed. The present study obtained lentiviral particles by high-speed centrifugation, and the lentiviral particle titer was 4 × 108 TU/mL after centrifugation. Thus, an optimized novel HIV-1 lentiviral vector was successfully constructed.
基金supported by the National Programs for High Technology Research and Development of China (2006AA10A204)the Gansu Key Technologies R&D Program(ZGS-052-A41-0006-03)the Programs for Director Fund of Lanzhou Veterinary Research Institute
文摘We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.
文摘The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.
文摘Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.
基金supported in part by the Special Fund for Agro-scientific Research in the Public Interest(201303016)the China Agriculture Research System(CARS-03)from the Ministry of Agriculture of China+3 种基金by the Project of International Scientific and Technical Cooperation(2013DFG31930)the National Key Technologies Research and Development Program of China(2012BAD19B04)the Breeding and Cultivation of Novel GM Varieties(2013ZX08002001)863 Program(2012AA101501)from the Ministry of Science and Technology of China
文摘The plasmid pGPDGFP under the control ofpgpdA promotor was used together with vector pAN7-1 containing the hygromycin resistance cassette to co-transform protoplasts of HG1, Fusarium graminearum from Hubei Province, China. Twelve out of 14 hygromycin-resistant transformants showed green signal under the UV light and contained one or several copies ofgfp, as indicated by Southern analysis of genomic DNA digested with different restriction enzymes and hybridized to the gfp probe. A single gfp copy transformant (HG1C5) was selected for further evaluation of 80 Chinese wheat cultivars or advanced lines. The results showed different resistance type to F. graminearum were observed. GFP signals observed in the rachis and adjacent spikes of 70 Chinese wheat lines such as Chuanchongzu 104 indicated both type I (host resistance to the initial infection by the fungus) and type II (resistance to the spread of FHB symptoms within an infected spike) were not observed. While other 10 lines showed type II resistance to F. graminearum with GFP signals only in inoculated spikelets. Development of the mycelium can be intuitively observed and the resistance of wheat to F. graminearum can be identified at 7 days post inoculation (dpi) in this way. The results showed no differences were evaluated between the transformed HG1C5 and the non-transgene artificial inoculation by SAS paired chi-square test and McNernar's test (P=-0.0625).
文摘The green fluorescent protein (GFP) from jellyfishAequorea victoria has unique superiority as a kind of biological label. GFP has been widely used in all fields of biology based on the recent studies on its structure, characteristic and mechanism of bioluninescence.
文摘In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.
文摘The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro , fluorescent VLPs could bind to CV 1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.