Purpose: To construct the enhanced yellow fluorescent protein (EYFP) vector carryinginterferon-y gene (ifn-γ) in order to provide an ideal reporter in the expression of ifn-γand location of protein in vitro and in v...Purpose: To construct the enhanced yellow fluorescent protein (EYFP) vector carryinginterferon-y gene (ifn-γ) in order to provide an ideal reporter in the expression of ifn-γand location of protein in vitro and in vivo.Method: According to the nucleotide sequence of ifn-y gene, a pair of oligonucleotideswas designed as primer whose two end contained nucleotide sequence of EcoR V and NotⅠ restriction endonuclease respectively. The gene encoding for inf-y was amplified usingPCR technique. After the PCR product was retrieved and purified, it was digested withEcoR V and Not Ⅰ restriction endonuclease, and then cloned into the plasmidpIRES-EYFP. The recombinant plasmid plRES-EYFPIFN-γwas identified by restrictionendonuclease enzyme analysis and DNA sequence analysis.Results: The ifn-γ was successfully amplified and verified by partial DNA sequenceanalysis. The recombinant plasmid was correctly screened.Conclusion: The EYFP expression vector carrying ifn-γgene was successfully established.This research work has formed a base for monitoring the ifn-y gene expression andprotein position in living cells.展开更多
A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated fr...A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated from spleen tissue pieces,which were cultured at 25℃ in Dulbecco's modiced Eagle medium/F12 medium(DMEM/F12,1:1)(pH7.2),supplemented with 20% fetal bovine serum,carboxymethyl chitosan,chondroitin sulfate,basic fibroblast growth factor(bFGF)and insulin-like growth factor-I(IGF-I).The cultured LYCS cells,in fibroblast shape,proliferated to 100% confluency 20 days later.Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P.crocea(6m+6sm+36t,NF=60).A LYCS cell line,with a population doubling time of 48.7 h at passage 60,has been established and subcultured to passage 70.Transgenic feasibility test demonstrated that positive green fluorescence protein(GFP)expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection.In conclusion,a continuous foreign gene trans-fection feasible LYCS cell line has been established successfully.The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.展开更多
Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalia...Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalian cell expression vector for CD11b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid of pCD11b-CFP. Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a target cell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. Then CHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scanning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed under a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were obtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-1CAM-1 cells was increased markedly by treatment with PMA, suggesting the translocation of GD11b-CFP and YFP-CD18 to the plasma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild type Mac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cells may be useful for the study of Mac-1 by FRET and for other purposes.展开更多
Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affe...Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.展开更多
A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activa...A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.展开更多
A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of...A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.展开更多
基金This paper is granted by National Nature Science Foudation of China(No.39700153)Natural Science Foundation of Guangdong Province(No.970082)
文摘Purpose: To construct the enhanced yellow fluorescent protein (EYFP) vector carryinginterferon-y gene (ifn-γ) in order to provide an ideal reporter in the expression of ifn-γand location of protein in vitro and in vivo.Method: According to the nucleotide sequence of ifn-y gene, a pair of oligonucleotideswas designed as primer whose two end contained nucleotide sequence of EcoR V and NotⅠ restriction endonuclease respectively. The gene encoding for inf-y was amplified usingPCR technique. After the PCR product was retrieved and purified, it was digested withEcoR V and Not Ⅰ restriction endonuclease, and then cloned into the plasmidpIRES-EYFP. The recombinant plasmid plRES-EYFPIFN-γwas identified by restrictionendonuclease enzyme analysis and DNA sequence analysis.Results: The ifn-γ was successfully amplified and verified by partial DNA sequenceanalysis. The recombinant plasmid was correctly screened.Conclusion: The EYFP expression vector carrying ifn-γgene was successfully established.This research work has formed a base for monitoring the ifn-y gene expression andprotein position in living cells.
基金supported by grants from the National High Technology Research and Development Program ('863' Program) of China (Grant Nos. 2006AA10A401 and 2006AA09Z406)
文摘A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated from spleen tissue pieces,which were cultured at 25℃ in Dulbecco's modiced Eagle medium/F12 medium(DMEM/F12,1:1)(pH7.2),supplemented with 20% fetal bovine serum,carboxymethyl chitosan,chondroitin sulfate,basic fibroblast growth factor(bFGF)and insulin-like growth factor-I(IGF-I).The cultured LYCS cells,in fibroblast shape,proliferated to 100% confluency 20 days later.Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P.crocea(6m+6sm+36t,NF=60).A LYCS cell line,with a population doubling time of 48.7 h at passage 60,has been established and subcultured to passage 70.Transgenic feasibility test demonstrated that positive green fluorescence protein(GFP)expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection.In conclusion,a continuous foreign gene trans-fection feasible LYCS cell line has been established successfully.The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.
基金Supported by National Natural Science Foundation of China (No.30000068, 39730210)
文摘Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalian cell expression vector for CD11b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid of pCD11b-CFP. Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a target cell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. Then CHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scanning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed under a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were obtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-1CAM-1 cells was increased markedly by treatment with PMA, suggesting the translocation of GD11b-CFP and YFP-CD18 to the plasma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild type Mac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cells may be useful for the study of Mac-1 by FRET and for other purposes.
基金financed by the National Natural Science Foundation of China (30900972, 31572111)the Special Found for Agro-scientific Research in the Public Interest, China (201203076-06)the Graduate Innovative Projects of Hunan Province, China (CX2013B290)
文摘Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.
基金the Start- up Fund for Returned Overseas Scholars from Northeast Normal U niversity,National ScienceFund for Distinguished Young Scholars (No. 30 32 5 0 11) ,Distinguished Young Scholars Fund of Jilin Province(No.2 0 0 30 112 ) ,Excellent Young Teachers
文摘A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.
文摘A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.
