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
Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by...Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase展开更多
Fluorescent Proteins(FP)can be applied as biomarkers and biosensors in the industrial and medical fields,but their large-scale use,especially for new industrial applications,is limited due to their low stability.Hence...Fluorescent Proteins(FP)can be applied as biomarkers and biosensors in the industrial and medical fields,but their large-scale use,especially for new industrial applications,is limited due to their low stability.Hence,the discovery of additives capable of preserving the activity of FP at room temperature and under stress conditions can help to expand and facilitate their commercial use.With this goal,we evaluated the application of 1-alkyl-3-methylimidazolium chloride-based([C_(n)mim]Cl)ionic liquids(ILs)as additives to preserve the activity of Enhanced Green Fluorescent Protein(EGFP)at different storage times and under unfavorable conditions.All[C_(n)mim]Cl solutions(at 0.100 mol L^(-1))were able to preserve EGFP fluorescence for longer than the phosphatesaline buffer(PBS)and NaCl solutions,increasing its fluorescence manifestation from 1 to 3 months.[C_(n)mim]Cl with shorter to medium cationic alkyl chains were the most effective in preserving EGFP fluorescence.[C_(n)mim]Cl also protected EGFP activity in the presence of the surfactant SDS,the acid guanidine hydrochloride,and H_(2)O_(2).Therefore,[C_(n)mim]Cl can be added to aqueous solutions to preserve EGFP fluorescence activity at room temperature for longer storage times and to reduce the negative impact of denaturing agents on EGFP.Therefore,there is a massive potential for the application of ILs as additives to preserve FP in the long-term without refrigeration and under unfavorable conditions,and this is fundamental to enable expansion of FP in industrial and commercial applications.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
Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for l...Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.展开更多
The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in ...The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.展开更多
While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs)...While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs),which are critical components of epigenetic regulation.lncRNAs are emerging as critical regulators of gene expression and genomic stability.However,it remains largely unknown about how lncRNAs are regulated.Here,we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion.Specifically,we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase(MODC)to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein,designated as dBiFP,and show that the dBiFP protein is highly destabilized,compared with the commonly-used eGFP protein.We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs.Therefore,our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs,synthetic regulatory RNA molecules,RNA binding proteins,and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.展开更多
We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy...We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases,neurotrauma,and stroke.In this study,using a mini pig model of spinal cord injury,we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy.Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector(Ad5/35 F)that carried an enhanced green fluorescent protein(EGFP)reporter gene in the plastic blood bag.The day after blood donation,the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate.A week after gene-modified leucoconcentrate therapy,fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury.In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed.In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes.Thus,the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions.This paper presents a proof-of-concept of simple,safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.5)on May 27,2014.展开更多
Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BM...Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy’s examination was to evaluate the results of the transduction,flow cytometer’s analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer’s analysis,the mean efficiency of the transduction with EGFP was（42.8±6.1）% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP2.Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2,and moreover,the expression had not attenuated over time.展开更多
Some studies indicate that adipose derived stem cells(ADSCs)can differentiate into adipogenic,chondrogenic,myogenic,and osteogenic cells in vitro.However,whether ADSCs can be induced to differentiate into neural cells...Some studies indicate that adipose derived stem cells(ADSCs)can differentiate into adipogenic,chondrogenic,myogenic,and osteogenic cells in vitro.However,whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated.In this study,the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene,and then the cells were induced for neural differentiation.The morphology of those ADSCs began to change within two days which developed into characteristics of round cell bodies with several branching extensions,concomitantly expressing EGFP fluorescence.Approximately 60% of the total cell populations were bipolar or multipolar in shape.Some of them appeared to make contact with their neighboring cells.RT-PCR,Western blot and Immunocytochemistry revealed that the expression levels of the markers of neurons and oligodendrocytes such as MAP2,NF-70,Neu N and RIP upon neural induction were increased,but the expression of the special marker of astrocytes,GFAP,was undetectable until 96h after induction when a small signal was observed.It was concluded that the ADSCs transfected with EGFP possessed the ability to undergo morphologic and phenotypic changes consistent with neural differentiation in vitro.It suggests that these cells might provide an ideal source for further stem cell research with possible therapeutic application for spinal cord injury.展开更多
Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( ...Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.展开更多
Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protopl...Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protoplasts of M. grisea. A library of 1077 transformants resistant to hygromycin B was generated. Of which, 448 transformants were found to express eGFP gene in different structures of M. grisea. Three transformants grew slowly, 5 transformants decreased in conidiation and 7 transformants reduced in pathogenicity greatly among these 448 transformants. Eleven transformants were checked by genomic southern blot randomly, and 9 of which were single-copy insertions. The promoter trapping technique has been applied successfully in M. grisea and can be used as a tool for functional genomic analysis.展开更多
Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of pro...Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer (TASMGT) mice were then mated with normal females at different stages of sexual maturity (6, 12, and 24 wk). The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group (15 μL gene suspension injected into each testis) and a high-dose group (30 μL suspensions injected) at the three stages of female sexual maturity tested were 11.76% (2/17), 14.29% (3/21), and 11.11% (2/18), and 5% (1/20), 5.56% (1/18), and 0 (0/17), respectively. The average integration rates for these two dose groups were 12.5% (7/56) and 3.64% (2/55), respectively, which was a significant difference (P<0.05). Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.展开更多
Through PCR amplification, 5' flanking region and partial open reading frame(ORF) of gene of Nile tilapia(Oreochromis niloticus) was cloned by PCR amplification. Sequence analysis showed that no difference was fou...Through PCR amplification, 5' flanking region and partial open reading frame(ORF) of gene of Nile tilapia(Oreochromis niloticus) was cloned by PCR amplification. Sequence analysis showed that no difference was found in known functional regions. This study was to construct and identify the mammalian expression vector of pEGFP-β-actin and to detect whether it could express in HEK 293T cell line. pEGFP-β-actin was transfected into HEK 293T cells with Lipofectamine 2000. The results showed that correct construction of recombinant pEGFP-β-actin has been shown by restriction enzyme digestion. The expression of gene in HEK 293T cells could be observed under microfluoroscope. pEGFP-β-actin could repress EGFP protein in HEK 293T cells. The results showed that β-actin gene promoter possessed effective transcription activities in eukaryotic cells. The work laid foundations for further study on the gene engineering and autotransgenic tilapia.展开更多
Microtubule cytoskeleton plays an important role in cell division,differentiation,cell morphology decision,introcellular material transportation and other physiological activities.Performing those function depends on ...Microtubule cytoskeleton plays an important role in cell division,differentiation,cell morphology decision,introcellular material transportation and other physiological activities.Performing those function depends on tubulin,microtubule-associated proteins(MAPs),correlated kinase and phosphate(ester) enzyme.In this article,GFP-TUA-6 and TUA-6-GFP gene were transferred into tobacco cotyledon mediated by Agrobacterium tumefaciens.Transformed lines were generated by the optimized genetic transformation system.It was proved that GFP-TUA-6 and TUA-6-GFP gene had been integrated into the tobacco genome by PCR detection and Southern blot analysis.The observation of laser scanning confocal microscope showed that GFP-TUA-6 protein was mainly located in cortical microtubules,while TUA-6-GFP protein was mainly located in cytoplasm and cortical microtubules,which provide a fundament for studying the dynamics activity of microtubule in cell morphogenesis.展开更多
This review is focused on using computer image analysis as a means of objective and quantitative characterizing optical images of the macroscopic (e.g. microbial colonies) and the microscopic (e.g. single cell) object...This review is focused on using computer image analysis as a means of objective and quantitative characterizing optical images of the macroscopic (e.g. microbial colonies) and the microscopic (e.g. single cell) objects in the microbiological research. This is the way of making many visual inspection assays more objective and less time and labor consuming. Also, it can provide new visually inaccessible information on relation between some optical parameters and various biological features of the microbial cul-tures. Of special interest is application of image analysis in fluorescence microscopy as it opens new ways of using fluorescence based methodology for single microbial cell studies. Examples of using image analysis in the studies of both the macroscopic and the microscopic microbiological objects obtained by various imaging techniques are presented and discussed.展开更多
The discovery of fluorescent proteins has inspired a lot of interest to develop synthetic polymers with similar structures and functionalities.In this review article,we first give a brief introduction for the discover...The discovery of fluorescent proteins has inspired a lot of interest to develop synthetic polymers with similar structures and functionalities.In this review article,we first give a brief introduction for the discovery,the structural characteristics and optical properties of fluorescent proteins,using green fluorescent protein(GFP)as an example.In the body part of article,we summarize recent advances in molecular design,synthesis and processing of bioinspired fluorescent polymers with unique optical properties such as high fluorescence quantum yield and robust photostability.Several major approaches highlighted here include the recombination of organic chromophores and proteins,and synthesis of the bioinspired polymers containing a single fluorophore in each polymer chain.The optical properties and applications of some representative bioinspired fluorescent polymers in bioimaging,sensing and imaging-guided therapy are also elucidated.展开更多
Current biological control methods to prevent pre-harvest aflatoxin contamination of corn, cottonseed, and ground and tree nuts involve field inoculation of non-aflatoxigenic Aspergillus flavus. To date, the efficacy ...Current biological control methods to prevent pre-harvest aflatoxin contamination of corn, cottonseed, and ground and tree nuts involve field inoculation of non-aflatoxigenic Aspergillus flavus. To date, the efficacy of this approach requires annual reapplication of the biocontrol agent. The reason for this requirement is uncertain. To track the dispersal and test the longevity of these strains, we prepared fluorescent biocontrol strains by incorporating into them the gene expressing the enhanced green fluorescent protein (eGFP). We first investigated the effects of eGFP transformation on the ability of the fluorescent fungus to compete with its non-fluorescent homolog, and then with other heterologous non-aflatoxigenic strains as well as with aflatoxigenic isolates. Our findings indicate that, in these studies, detection of fluorescence was variable, with some fluorescent strains exhibiting enhanced growth and sporulation post-transformation. In our tests, not all transformed strains proved to be good candidates for tracking because their fluorescence was reduced over the course of our study. Most of the transformed strains retained fluorescence and showed robust colony growth in an artificial competitor environment;therefore, they should be suited for further trial under more natural settings. Our ultimate objective is to determine if out-crossing between biocontrol strains and native field populations is occurring in a natural setting.展开更多
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.
基金This work was supported by grantsfromThe Int .Cooperation Projectfor National &Abroad Lab.of the National Natural Sciences Foundation ofChina(2002008) and The Science &Technology Foundation of Liaoningprovince (20022140)
文摘Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase
基金the project“Understanding the molecular interactions between ionic liquids and biopharmaceuticals:the key for a proper design of downstream processing”,2018/50009–8,co-funded by FAPESP(S~ao Paulo Research Foundation,Brazil)and Australian Technology Network of Universities(ATN)and projects FAPESP 2014/19793–3,2014/16424–7 and 2018/25511–1.N.V.Veríssimo(2016/07529–5,2018/06576–5,and 2020/14144–8)C.F.Saponi(2018/01858–2 and 2018/20833–0)acknowledge scholarships financial support from FAPESP.N.V.Veríssimo,J.F.B.Pereira also acknowledge the funding from CNPq,CAPES(001),CAPES-PROEX.CIEPQPF is supported by the FCT through the projects UIDB/EQU/00102/2020 and UIDP/EQU/00102/2020.
文摘Fluorescent Proteins(FP)can be applied as biomarkers and biosensors in the industrial and medical fields,but their large-scale use,especially for new industrial applications,is limited due to their low stability.Hence,the discovery of additives capable of preserving the activity of FP at room temperature and under stress conditions can help to expand and facilitate their commercial use.With this goal,we evaluated the application of 1-alkyl-3-methylimidazolium chloride-based([C_(n)mim]Cl)ionic liquids(ILs)as additives to preserve the activity of Enhanced Green Fluorescent Protein(EGFP)at different storage times and under unfavorable conditions.All[C_(n)mim]Cl solutions(at 0.100 mol L^(-1))were able to preserve EGFP fluorescence for longer than the phosphatesaline buffer(PBS)and NaCl solutions,increasing its fluorescence manifestation from 1 to 3 months.[C_(n)mim]Cl with shorter to medium cationic alkyl chains were the most effective in preserving EGFP fluorescence.[C_(n)mim]Cl also protected EGFP activity in the presence of the surfactant SDS,the acid guanidine hydrochloride,and H_(2)O_(2).Therefore,[C_(n)mim]Cl can be added to aqueous solutions to preserve EGFP fluorescence activity at room temperature for longer storage times and to reduce the negative impact of denaturing agents on EGFP.Therefore,there is a massive potential for the application of ILs as additives to preserve FP in the long-term without refrigeration and under unfavorable conditions,and this is fundamental to enable expansion of FP in industrial and commercial applications.
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supported by the Zhejiang Foundation Public Welfare Research Project(Authorization No.:LGF19B060006)。
文摘Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.
基金This work was supported by the Research Council of NorrbottenIn-novationsbron and Langmanska foretagarfonden.J.H.was supported by the Swedish Research Council(No.K2011-65X-3031-01-6)the County Council of Vastra Gotaland(ALF).
文摘The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.
基金The reported work was supported in part by research grants from the National Institutes of Health(AT004418,DE020140 to TCH and RRR)the US Department of Defense(OR130096 to JMW)+5 种基金the Scoliosis Research Society(TCH and MJL)the National Key Research and Development Program of China(2016YFC1000803 and 2011CB707906 to TCH)the National Natural Science Foundation of China(#81201916 to XW)ZZ was a recipient of protectorate fellowship from China Scholarship CouncilThis project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430.
文摘While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs),which are critical components of epigenetic regulation.lncRNAs are emerging as critical regulators of gene expression and genomic stability.However,it remains largely unknown about how lncRNAs are regulated.Here,we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion.Specifically,we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase(MODC)to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein,designated as dBiFP,and show that the dBiFP protein is highly destabilized,compared with the commonly-used eGFP protein.We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs.Therefore,our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs,synthetic regulatory RNA molecules,RNA binding proteins,and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.
基金the Russian Science Foundation(No.16-15-00010to RRI)the Russian Government Program of Competitive Growth of Kazan Federal University。
文摘We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases,neurotrauma,and stroke.In this study,using a mini pig model of spinal cord injury,we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy.Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector(Ad5/35 F)that carried an enhanced green fluorescent protein(EGFP)reporter gene in the plastic blood bag.The day after blood donation,the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate.A week after gene-modified leucoconcentrate therapy,fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury.In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed.In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes.Thus,the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions.This paper presents a proof-of-concept of simple,safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.5)on May 27,2014.
基金This work was supported by the National Natural Science Foundation of China(No.30400163).
文摘Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy’s examination was to evaluate the results of the transduction,flow cytometer’s analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer’s analysis,the mean efficiency of the transduction with EGFP was（42.8±6.1）% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP2.Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2,and moreover,the expression had not attenuated over time.
基金supported by the "Eleventh Five-Year" plan to support the National Science and Technology key project(No.2007BAI04B07)a grant from National Natural Sciences Foundation of China(No.30772206)
文摘Some studies indicate that adipose derived stem cells(ADSCs)can differentiate into adipogenic,chondrogenic,myogenic,and osteogenic cells in vitro.However,whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated.In this study,the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene,and then the cells were induced for neural differentiation.The morphology of those ADSCs began to change within two days which developed into characteristics of round cell bodies with several branching extensions,concomitantly expressing EGFP fluorescence.Approximately 60% of the total cell populations were bipolar or multipolar in shape.Some of them appeared to make contact with their neighboring cells.RT-PCR,Western blot and Immunocytochemistry revealed that the expression levels of the markers of neurons and oligodendrocytes such as MAP2,NF-70,Neu N and RIP upon neural induction were increased,but the expression of the special marker of astrocytes,GFAP,was undetectable until 96h after induction when a small signal was observed.It was concluded that the ADSCs transfected with EGFP possessed the ability to undergo morphologic and phenotypic changes consistent with neural differentiation in vitro.It suggests that these cells might provide an ideal source for further stem cell research with possible therapeutic application for spinal cord injury.
基金Scientific Research Project for High Schools of the Educational Department of Liaoning Province,China(No.2008643)
文摘Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.
基金Project supported by the National Natural Science Foundation of China (Nos. 30270049 and 30470064) and the Hi-Tech Research and Development Program (863) of China (No. 2002AA245041)
文摘Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protoplasts of M. grisea. A library of 1077 transformants resistant to hygromycin B was generated. Of which, 448 transformants were found to express eGFP gene in different structures of M. grisea. Three transformants grew slowly, 5 transformants decreased in conidiation and 7 transformants reduced in pathogenicity greatly among these 448 transformants. Eleven transformants were checked by genomic southern blot randomly, and 9 of which were single-copy insertions. The promoter trapping technique has been applied successfully in M. grisea and can be used as a tool for functional genomic analysis.
基金supported by the National Transgenic Breeding Project (2008ZX08010-004)the National Natural Science Foundation of China (30830080)+1 种基金the National 973 Program of China (G2006CB102105,2009CB941604)the National 863 Program of China (20060110Z1039, 2008AA10Z143)
文摘Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer (TASMGT) mice were then mated with normal females at different stages of sexual maturity (6, 12, and 24 wk). The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group (15 μL gene suspension injected into each testis) and a high-dose group (30 μL suspensions injected) at the three stages of female sexual maturity tested were 11.76% (2/17), 14.29% (3/21), and 11.11% (2/18), and 5% (1/20), 5.56% (1/18), and 0 (0/17), respectively. The average integration rates for these two dose groups were 12.5% (7/56) and 3.64% (2/55), respectively, which was a significant difference (P<0.05). Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.
基金Supported by China Agriculture Research System(CARS-49)Fujian Seed Industry Innovation and Industrialization(2011FJZY)
文摘Through PCR amplification, 5' flanking region and partial open reading frame(ORF) of gene of Nile tilapia(Oreochromis niloticus) was cloned by PCR amplification. Sequence analysis showed that no difference was found in known functional regions. This study was to construct and identify the mammalian expression vector of pEGFP-β-actin and to detect whether it could express in HEK 293T cell line. pEGFP-β-actin was transfected into HEK 293T cells with Lipofectamine 2000. The results showed that correct construction of recombinant pEGFP-β-actin has been shown by restriction enzyme digestion. The expression of gene in HEK 293T cells could be observed under microfluoroscope. pEGFP-β-actin could repress EGFP protein in HEK 293T cells. The results showed that β-actin gene promoter possessed effective transcription activities in eukaryotic cells. The work laid foundations for further study on the gene engineering and autotransgenic tilapia.
基金supported by The Henan Province Science and Technology Development Program in China(grant no.092102310044)The Ministry of Education Innovative Talent Supporting Program of Henan province in China(grant no.2010HASTIT019)
文摘Microtubule cytoskeleton plays an important role in cell division,differentiation,cell morphology decision,introcellular material transportation and other physiological activities.Performing those function depends on tubulin,microtubule-associated proteins(MAPs),correlated kinase and phosphate(ester) enzyme.In this article,GFP-TUA-6 and TUA-6-GFP gene were transferred into tobacco cotyledon mediated by Agrobacterium tumefaciens.Transformed lines were generated by the optimized genetic transformation system.It was proved that GFP-TUA-6 and TUA-6-GFP gene had been integrated into the tobacco genome by PCR detection and Southern blot analysis.The observation of laser scanning confocal microscope showed that GFP-TUA-6 protein was mainly located in cortical microtubules,while TUA-6-GFP protein was mainly located in cytoplasm and cortical microtubules,which provide a fundament for studying the dynamics activity of microtubule in cell morphogenesis.
文摘This review is focused on using computer image analysis as a means of objective and quantitative characterizing optical images of the macroscopic (e.g. microbial colonies) and the microscopic (e.g. single cell) objects in the microbiological research. This is the way of making many visual inspection assays more objective and less time and labor consuming. Also, it can provide new visually inaccessible information on relation between some optical parameters and various biological features of the microbial cul-tures. Of special interest is application of image analysis in fluorescence microscopy as it opens new ways of using fluorescence based methodology for single microbial cell studies. Examples of using image analysis in the studies of both the macroscopic and the microscopic microbiological objects obtained by various imaging techniques are presented and discussed.
基金We acknowledge the financial support by the University Development Fund-Research Start-up Fund(UDF01001806)from the Chinese University of Hong Kong,Shenzhen.H.X.and W.Z.thank the financial support of Ph.D.Scholarship from the Chinese University of Hong Kong,Shenzhen.
文摘The discovery of fluorescent proteins has inspired a lot of interest to develop synthetic polymers with similar structures and functionalities.In this review article,we first give a brief introduction for the discovery,the structural characteristics and optical properties of fluorescent proteins,using green fluorescent protein(GFP)as an example.In the body part of article,we summarize recent advances in molecular design,synthesis and processing of bioinspired fluorescent polymers with unique optical properties such as high fluorescence quantum yield and robust photostability.Several major approaches highlighted here include the recombination of organic chromophores and proteins,and synthesis of the bioinspired polymers containing a single fluorophore in each polymer chain.The optical properties and applications of some representative bioinspired fluorescent polymers in bioimaging,sensing and imaging-guided therapy are also elucidated.
文摘Current biological control methods to prevent pre-harvest aflatoxin contamination of corn, cottonseed, and ground and tree nuts involve field inoculation of non-aflatoxigenic Aspergillus flavus. To date, the efficacy of this approach requires annual reapplication of the biocontrol agent. The reason for this requirement is uncertain. To track the dispersal and test the longevity of these strains, we prepared fluorescent biocontrol strains by incorporating into them the gene expressing the enhanced green fluorescent protein (eGFP). We first investigated the effects of eGFP transformation on the ability of the fluorescent fungus to compete with its non-fluorescent homolog, and then with other heterologous non-aflatoxigenic strains as well as with aflatoxigenic isolates. Our findings indicate that, in these studies, detection of fluorescence was variable, with some fluorescent strains exhibiting enhanced growth and sporulation post-transformation. In our tests, not all transformed strains proved to be good candidates for tracking because their fluorescence was reduced over the course of our study. Most of the transformed strains retained fluorescence and showed robust colony growth in an artificial competitor environment;therefore, they should be suited for further trial under more natural settings. Our ultimate objective is to determine if out-crossing between biocontrol strains and native field populations is occurring in a natural setting.
