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
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
The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and e...The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.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
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
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 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This work was supported by the National Natural Science Foundation of China[31870040]the National Key Research and Development Project(2020YFA0907804,2020YFA0907304)+1 种基金the“111”Project of China[B18022]the Fundamental Research Funds for the Central Universities[22221818014],and the Open Project Funding of the State Key Laboratory of Bioreactor Engineering.
文摘The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.
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 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.
