AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR).
CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutio...CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science.Here,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant species.We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts.The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors.In systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency.Plants with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection.Moreover,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable mutations.Because many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.展开更多
[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector fo...[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.展开更多
[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene seque...[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.展开更多
The initial phase of tomato is critical to the infestation of insect vectors of viruses. Therefore, this study aimed to test the use of yellow card traps around the crop to manage insect vectors of viruses and test th...The initial phase of tomato is critical to the infestation of insect vectors of viruses. Therefore, this study aimed to test the use of yellow card traps around the crop to manage insect vectors of viruses and test the best density of traps/tomato plants. Yellow card traps were placed on the border of the crop plot to capture adult insect vectors. Density of trap/tomato plant was assessed in 10 blocks at the following levels： 1/25; 1/50; 1/75; 1/100; 1/125; 1/150. The monitoring was carded out in 1% of the crop during 60 days in 2011 and 2012 crop. The evaluated systems were Conventional and Phytosanitary Pest Management （PPM）. During 2011 season the Conventional system received 14 insecticide applications whereas only 6 insecticide applications were made on the PPM, representing a reduction of 133%. In 2012, the crop under Conventional system was subjected to 15 applications of insecticides, over 8 on PPM, with a reduction of 87.5%. The PPM allowed a 90% reduction in application cost for this insects, obtaining a reduction of R$1,345.00/ha. The highest density was 60 plants/trap. We can conclude that the yellow card traps in tomato crop decreased infestations of insect vectors of viruses.展开更多
[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstep...[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.展开更多
BACKGROUND： The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression. OBJECTIVE： This study was designed to transfect a human brain glioma cell line with recombina...BACKGROUND： The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression. OBJECTIVE： This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 （rVV-IL-2） gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN： Experimental observation. SETTING： Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS： The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. METHODS： At passage day l, Veto cells were amplified l ; 1 for virus and cells. A human brain glioma cell line was transfected using amplified Vaccinia viral vectors at varying multiplicities of infection （MOI）. At 2, 4, 6, 8, 12, and 24 hours post-transfection, superuatant was collected to determine by MTT assay IL-2 expression levels in IL-2 dependent cells. The transfected and non-transfected cells were divided into 4 groups, namely MOI1 ： 1, MOI 5 ： 1, MOI 10 ： 1, and control groups. MAIN OUTCOME MEASURES： IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS： IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups （P 〉 0.05）. CONCLUSION： Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.展开更多
Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested...Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested with BamHⅠ and XhoⅠ, respectively. Then the Smad6/Flag and Smad7/Flag gene segments obtained were cloned into plasmid pAAV-MCS respectively to construct the recombinant pAAV-Smad6/Flag and pAAV-Smad7/Flag plasmids. The resulting recombinant plasmids (pAAV-Smad6/Flag or pAAV-Smad7/Flag) or pAAV-LacZ plasmid were co-transfected into the HEK 293cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect mouse macrophages. The expressions of Smad6 and Smad7 in macrophages were detected by immunocytochemical staining and expression of b-galactosidase was evaluated by X-gal staining. Results: The recombinant AAV vector containing Smad6 or Smad7 genes was successfully constructed. More than 95% macrophage cells expressed X-gal and Smad6 and Smad7 genes at 72 h after infection. Conclusion: These results indicate that the genetically engineered macrophages can express Smad6 and Smad7 proteins effectively, laying the foundation for the studies of TGF-β-induced diseases in vivo and highlighting the feasibility of macrophage-based gene therapy.展开更多
The present study employed a rat model of T10 spinal cord transection. Western blot analyses revealed increased brain-dedved neurotrophic factor （BDNF） expression in spinal cord segments caudal to the transection si...The present study employed a rat model of T10 spinal cord transection. Western blot analyses revealed increased brain-dedved neurotrophic factor （BDNF） expression in spinal cord segments caudal to the transection site following injection of replication incompetent herpes simplex virus vector （HSV-BDNF） into the subarachnoid space. In addition, hindlimb locomotor functions were improved. In contrast, BDNF levels decreased following treatment with replication defective herpes simplex virus vector construct small interference BDNF （HSV-siBDNF）. Moreover, hindlimb locomotor functions gradually worsened. Compared with the replication incompetent herpes simplex virus vector control group, extracellular signal regulated kinasel/2 expression increased in the HSV-BDNF group on days 14 and 28 after spinal cord transection, but expression was reduced in the HSV-siBDNF group. These results suggested that BDNF plays an important role in neural plasticity via extracellular signal regulated kinasel/2 signaling pathway in a rat model of adult spina cord transection.展开更多
Exogenous neuropeptide Y has antiepileptic effects; however, the underlying mechanism and optimal administration method for neuropeptide Y are still unresolved. Previous studies have used intracerebroventricular injec...Exogenous neuropeptide Y has antiepileptic effects; however, the underlying mechanism and optimal administration method for neuropeptide Y are still unresolved. Previous studies have used intracerebroventricular injection of neuropeptide Y into animal models of epilepsy. In this study, a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene was injected into the lateral ventricle of rats, while the ipsilateral hippocampus was injected with kainic acid to establish the epileptic model. After transfection of neuropeptide Y gene, mossy fiber sprouting in the hippocampal CA3 region of epileptic rats was significantly suppressed, hippocampal synaptophysin （p38） mRNA and protein expression were inhibited, and epileptic seizures were reduced. These experimental findings indicate that a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene reduces mossy fiber sprouting and inhibits abnormal synaptophysin expression, thereby suppressing post-epileptic synaptic reconstruction.展开更多
基金Supported by In part by the 21st Century COE(Center Of Ex-cellence)Programs to Dr.Takenori Ochiaiby a Grant-in-Aid 18591453 to K.M from the Ministry of Education,Science,Sports and Culture of Japan
文摘AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR).
基金supported by grants from the National Key R&D Program of China(2022YFC2601000)the Natural Science Foundation of Zhejiang Province,China(no.LZ20C140004)the National Natural Science Foundation of China(no.31870142).
文摘CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science.Here,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant species.We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts.The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors.In systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency.Plants with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection.Moreover,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable mutations.Because many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.
基金Supported by Natural Science Foundation of Liaoning Province(20072122)Projects Funding of Liaoning Provincial Education Office(05L339)~~
文摘[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.
基金Supported by International Science and Technology Cooperation Program (2008DFA30560)Preliminary Research Special Foundation of 973 Program (2008CB117018)Scientific Research Project for High Level of Talents of Shihezi University (RCZX200732)~~
文摘[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.
文摘The initial phase of tomato is critical to the infestation of insect vectors of viruses. Therefore, this study aimed to test the use of yellow card traps around the crop to manage insect vectors of viruses and test the best density of traps/tomato plants. Yellow card traps were placed on the border of the crop plot to capture adult insect vectors. Density of trap/tomato plant was assessed in 10 blocks at the following levels： 1/25; 1/50; 1/75; 1/100; 1/125; 1/150. The monitoring was carded out in 1% of the crop during 60 days in 2011 and 2012 crop. The evaluated systems were Conventional and Phytosanitary Pest Management （PPM）. During 2011 season the Conventional system received 14 insecticide applications whereas only 6 insecticide applications were made on the PPM, representing a reduction of 133%. In 2012, the crop under Conventional system was subjected to 15 applications of insecticides, over 8 on PPM, with a reduction of 87.5%. The PPM allowed a 90% reduction in application cost for this insects, obtaining a reduction of R$1,345.00/ha. The highest density was 60 plants/trap. We can conclude that the yellow card traps in tomato crop decreased infestations of insect vectors of viruses.
文摘[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.
文摘BACKGROUND： The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression. OBJECTIVE： This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 （rVV-IL-2） gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN： Experimental observation. SETTING： Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS： The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. METHODS： At passage day l, Veto cells were amplified l ; 1 for virus and cells. A human brain glioma cell line was transfected using amplified Vaccinia viral vectors at varying multiplicities of infection （MOI）. At 2, 4, 6, 8, 12, and 24 hours post-transfection, superuatant was collected to determine by MTT assay IL-2 expression levels in IL-2 dependent cells. The transfected and non-transfected cells were divided into 4 groups, namely MOI1 ： 1, MOI 5 ： 1, MOI 10 ： 1, and control groups. MAIN OUTCOME MEASURES： IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS： IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups （P 〉 0.05）. CONCLUSION： Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.
文摘Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested with BamHⅠ and XhoⅠ, respectively. Then the Smad6/Flag and Smad7/Flag gene segments obtained were cloned into plasmid pAAV-MCS respectively to construct the recombinant pAAV-Smad6/Flag and pAAV-Smad7/Flag plasmids. The resulting recombinant plasmids (pAAV-Smad6/Flag or pAAV-Smad7/Flag) or pAAV-LacZ plasmid were co-transfected into the HEK 293cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect mouse macrophages. The expressions of Smad6 and Smad7 in macrophages were detected by immunocytochemical staining and expression of b-galactosidase was evaluated by X-gal staining. Results: The recombinant AAV vector containing Smad6 or Smad7 genes was successfully constructed. More than 95% macrophage cells expressed X-gal and Smad6 and Smad7 genes at 72 h after infection. Conclusion: These results indicate that the genetically engineered macrophages can express Smad6 and Smad7 proteins effectively, laying the foundation for the studies of TGF-β-induced diseases in vivo and highlighting the feasibility of macrophage-based gene therapy.
文摘The present study employed a rat model of T10 spinal cord transection. Western blot analyses revealed increased brain-dedved neurotrophic factor （BDNF） expression in spinal cord segments caudal to the transection site following injection of replication incompetent herpes simplex virus vector （HSV-BDNF） into the subarachnoid space. In addition, hindlimb locomotor functions were improved. In contrast, BDNF levels decreased following treatment with replication defective herpes simplex virus vector construct small interference BDNF （HSV-siBDNF）. Moreover, hindlimb locomotor functions gradually worsened. Compared with the replication incompetent herpes simplex virus vector control group, extracellular signal regulated kinasel/2 expression increased in the HSV-BDNF group on days 14 and 28 after spinal cord transection, but expression was reduced in the HSV-siBDNF group. These results suggested that BDNF plays an important role in neural plasticity via extracellular signal regulated kinasel/2 signaling pathway in a rat model of adult spina cord transection.
文摘Exogenous neuropeptide Y has antiepileptic effects; however, the underlying mechanism and optimal administration method for neuropeptide Y are still unresolved. Previous studies have used intracerebroventricular injection of neuropeptide Y into animal models of epilepsy. In this study, a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene was injected into the lateral ventricle of rats, while the ipsilateral hippocampus was injected with kainic acid to establish the epileptic model. After transfection of neuropeptide Y gene, mossy fiber sprouting in the hippocampal CA3 region of epileptic rats was significantly suppressed, hippocampal synaptophysin （p38） mRNA and protein expression were inhibited, and epileptic seizures were reduced. These experimental findings indicate that a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene reduces mossy fiber sprouting and inhibits abnormal synaptophysin expression, thereby suppressing post-epileptic synaptic reconstruction.
