【目的】利用P2C可以定向进入卵巢以及Gal4蛋白可与UAS序列稳定结合的特点,在中华按蚊Anopheles sinensis中建立高效的非胚胎期外源DNA投递技术系统。【方法】注射P2C-Gal4-DsRed重组蛋白至吸血后20 h时的中华按蚊雌成蚊腹部,通过冰冻...【目的】利用P2C可以定向进入卵巢以及Gal4蛋白可与UAS序列稳定结合的特点,在中华按蚊Anopheles sinensis中建立高效的非胚胎期外源DNA投递技术系统。【方法】注射P2C-Gal4-DsRed重组蛋白至吸血后20 h时的中华按蚊雌成蚊腹部,通过冰冻切片荧光观察和Western blot检测分析重组蛋白P2C-Gal4-DsRed在卵巢中的投递效率;制备P2C-Gal4 DNA BINDING重组蛋白,构建包含12×UAS重复基序的转基因质粒和辅助质粒,通过电泳迁移实验分析重组蛋白P2C-Gal4 DNA BINDING和12×UAS重复基序间的体外结合;分别将体外孵育的P2C-Gal4 DNA BINDING+辅助质粒ITF36-12×UAS和P2C-Gal4 DNA BINDING+转基因质粒ITF2-12×UAS afm复合物注射入吸血后20 h时的中华按蚊雌成蚊腹部,于血餐后40 h时提取其卵巢组织DNA,并通过特异性引物PCR扩增和测序分析外源DNA在活体中的投递情况。【结果】100%注射P2C-Gal4-DsRed的中华按蚊雌成蚊卵巢在绿色滤光片下呈现明显的红色荧光,表明P2C-Gal4-DsRed重组蛋白能够被高效地导入雌成蚊卵巢中;P2C-Gal4 DNA BINDING重组蛋白能够与12×UAS重复基序以及含有该重复基序片段的质粒稳定结合;分别有91%和93%的注射了P2C-Gal4 DNA BINDING+ITF36-12×UAS和P2C-Gal4 DNA BINDING+ITF2-12×UAS afm的雌成蚊卵巢组织中能够检测到外源DNA片段。【结论】在中华按蚊中成功建立了基于P2C卵巢导向肽和Gal4-12×UAS重复基序结合特性的外源DNA投递技术体系;通过此技术平台能够便捷、快速和高效地实现质粒等DNA分子在中华按蚊卵巢中的投递,这为进一步简化转基因、过表达及基因敲入等遗传操作奠定了基础。展开更多
The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into ...The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.展开更多
We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding ...We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding to 236 human genes have been established. In all, 51 lines are capable of eliciting a phenotype suggesting that the human genes are functional. These heterologous genes are functionally relevant as we have found a similar mutant phenotype caused either by a dominant negative mutant form of the human ribosomal protein L8 gene or by RNAi downregulation of the Drosophila RPL8. Significantly, the Drosophila RPL8 mutant can be rescued by wild-type human RPL8. We also provide genetic evidence that Drosophila RPL8 is a new member of the insulin signaling pathway. In summary, the functions of many human genes appear to be highly conserved, and the ability to identify them in Drosophila represents a powerful genetic tool for large-scale analysis of human transcripts in vivo.展开更多
Proteins containing an expanded polyglutamine tract are neurotoxins. The expanded polyglutamine proteins influence a variety of cellular functions. In Drosophila the GMR-Gal4/UAS expression system has been widely used...Proteins containing an expanded polyglutamine tract are neurotoxins. The expanded polyglutamine proteins influence a variety of cellular functions. In Drosophila the GMR-Gal4/UAS expression system has been widely used in an eye-based model to study human neurodegenerative diseases. This system has facilitated the isolation and characterization of abundant Drosophilagenes that interact with the expanded polyglutamine proteins. We used the GMR-Gal4/UAS system to express three proteins containing an expanded polyglutamine tract, or an expanded polyglutamine tract alone. Doubling the dose of these proteins resulted in pupal lethality, indicating that these toxic proteins induced a sensitized condition that is prone to synthetic lethality. By using the GMR-Gal4/UAS system, we showed that a Drosophilagene interacts with three expanded polyglutamine proteins to induce a synthetic lethal phenotype. We further demonstrated that the synthetic lethality was mediated through the toxic expanded polyglutamine tract. Our study raises a possibility that conventional genetic screens may not recover synthetic lethal alleles, which are presumably stronger interacting alleles than the currently known modifiers of an expanded polyglutamine tract, due to synthetic lethality.展开更多
Mutant proteins containing an expanded polyglutamine tract induce cell death and cause neurodegenerative diseases. These toxic proteins interfere with a variety of physiological pathways, but the key interactions betw...Mutant proteins containing an expanded polyglutamine tract induce cell death and cause neurodegenerative diseases. These toxic proteins interfere with a variety of physiological pathways, but the key interactions between the toxins and cellular factors remain unclear. To model the diseases in Drosophila, the GMR-Gal4/UAS gene expression system has been used extensively, which operates in the eyes. By using the system, genome-wide studies have resulted in the isolation of functionally diverse groups of Drosophila genes that interact with the disease proteins. We previously reported that coexpressing the Drosophila Dikar gene and an expanded polyglutamine tract by GMR-Gal4/UAS induced a synthetic lethality. We carried out follow-up experiments to isolate additional synthetic lethal alleles. Our data provide evidence that synthetic lethality associated with expressing an expanded polyglutamine tract is more common than thought to be and could have escaped the conventional genetic screens. Our results also suggest that 1) the gene expression system is leaky, allowing expression outside of the primary target eye cell types;2) expressing an expanded polyglutamine tract is extremely toxic to cells;and 3) combining the leaky expression and the toxicity results in a lethal-prone condition. Thus, genetic modifications to the disease proteins’ acute toxicity could frequently lead to synthetic lethality. However, synthetic lethal alleles are excluded from most conventional screens, necessitating alternative approaches such as a two-step method used in this study to isolate the modifiers. Since synthetic lethality reflects essential genetic buffering networks, studying these alleles may hold the keys to identify the critical interactions in the disease development between the toxic proteins and the physiological pathways.展开更多
Alzheimer’s disease(AD)is one of the most common forms of dementia.Cognitive dysfunction and memory loss are the two main clinical symptoms of AD.Drosophila melanogaster models of AD,which are based on overexpression...Alzheimer’s disease(AD)is one of the most common forms of dementia.Cognitive dysfunction and memory loss are the two main clinical symptoms of AD.Drosophila melanogaster models of AD,which are based on overexpression of human amyloidβ(Aβ)or human tau(hTau)protein,have been used to study the mechanism underlying AD and to screen potential therapeutic compounds.Drugs that are currently available for AD provide only symptomatic relief.Huge unmet medical needs exists to slow,stop,or reverse the progression of AD.Thymoquinone(TQ)is an active ingredient isolated from Nigella sativa(NS)and possesses various pharmacological activities,and it is also a potential neuropharmacological agent.The current study was performed to investigate the effect of dietary administration of TQ at concentrations of 12.5μM and 25μM for 15 and 30 days on biochemical and behavioral parameters,gene,and protein expression of hTau,using Drosophila models of AD.Transgenic Drosophila models exhibiting pan-neuronal and eye-specific expression of hTau were generated using the GAL4/UAS system.Treatment with TQ at both concentrations resulted in a significant increase in behavioral activity,a significant reduction in the amount of reactive oxygen species(ROS),and restoration of depleted superoxide dismutase(SOD)and acetylcholine esterase(AChE)activities.A significant decrease in gene and protein expression of hTau was also observed at the molecular level for both concentrations of TQ.Therefore,TQ has the potential to be investigated as a potential therapeutic phytochemical for the treatment of AD in future studies.展开更多
The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products.In sericulture,only the first filial generation(F1)hybrid eggs produced by cross-breedin...The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products.In sericulture,only the first filial generation(F1)hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms,but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs.To address this issue,we developed a safe and efficient strategy using the GAL4/Upstream activating sequence(UAS)system,the FLP/flippase recognition target(FRT)system,and the gonad-specific expression gene promoters(RSHP1p and Nanosp)for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms.We established 2 types of activator strains,R1p::GAL4-Gr and Nsp::GAL4-Gr,containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp,respectively,and 1 type of effector strain,UAS::FLP-Rg,containing the UAS-linked FLP gene expression cassette.The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%,whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73%to 80.3%.Additionally,we identified a gene,sw11114,that is expressed in both testis and ovary of Bombyx mori,and can be used to establish novel gonad-specific expression systems in transgenic silkworms.This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.展开更多
文摘【目的】利用P2C可以定向进入卵巢以及Gal4蛋白可与UAS序列稳定结合的特点,在中华按蚊Anopheles sinensis中建立高效的非胚胎期外源DNA投递技术系统。【方法】注射P2C-Gal4-DsRed重组蛋白至吸血后20 h时的中华按蚊雌成蚊腹部,通过冰冻切片荧光观察和Western blot检测分析重组蛋白P2C-Gal4-DsRed在卵巢中的投递效率;制备P2C-Gal4 DNA BINDING重组蛋白,构建包含12×UAS重复基序的转基因质粒和辅助质粒,通过电泳迁移实验分析重组蛋白P2C-Gal4 DNA BINDING和12×UAS重复基序间的体外结合;分别将体外孵育的P2C-Gal4 DNA BINDING+辅助质粒ITF36-12×UAS和P2C-Gal4 DNA BINDING+转基因质粒ITF2-12×UAS afm复合物注射入吸血后20 h时的中华按蚊雌成蚊腹部,于血餐后40 h时提取其卵巢组织DNA,并通过特异性引物PCR扩增和测序分析外源DNA在活体中的投递情况。【结果】100%注射P2C-Gal4-DsRed的中华按蚊雌成蚊卵巢在绿色滤光片下呈现明显的红色荧光,表明P2C-Gal4-DsRed重组蛋白能够被高效地导入雌成蚊卵巢中;P2C-Gal4 DNA BINDING重组蛋白能够与12×UAS重复基序以及含有该重复基序片段的质粒稳定结合;分别有91%和93%的注射了P2C-Gal4 DNA BINDING+ITF36-12×UAS和P2C-Gal4 DNA BINDING+ITF2-12×UAS afm的雌成蚊卵巢组织中能够检测到外源DNA片段。【结论】在中华按蚊中成功建立了基于P2C卵巢导向肽和Gal4-12×UAS重复基序结合特性的外源DNA投递技术体系;通过此技术平台能够便捷、快速和高效地实现质粒等DNA分子在中华按蚊卵巢中的投递,这为进一步简化转基因、过表达及基因敲入等遗传操作奠定了基础。
基金Supported by National High-tech Research and Development Program of China(No.2007AA021004)
文摘The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.
基金We are grateful to Xizhi Ma, Junnian Zhou, Tianhong Xu, Xu Liu, Xu Ding, Yang Liu, Ying Peng, Congwu Chi, Yiying Shang, Mingyao Ying, Sheng Ding, Lei Sun, Lei Tian, Huanhu Zhu, Hua Huang, Hongmei Li, and Xiaomo Wu for cDNA constructs and partial transgenic work, and Lihui Zhou （East China University of Science and Technology, China） for scanning electron microscopy. We thank Duc Nguyen （Yale University, USA） for critical reading and editing of this manuscript. This work is supported by grants from the National Natural Science Foundation of China （Grant Nos. 30030080, 39970408 and 30470840）, National Basic Research Program of China （973） （Grant No. 2006CB806700）.
文摘We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding to 236 human genes have been established. In all, 51 lines are capable of eliciting a phenotype suggesting that the human genes are functional. These heterologous genes are functionally relevant as we have found a similar mutant phenotype caused either by a dominant negative mutant form of the human ribosomal protein L8 gene or by RNAi downregulation of the Drosophila RPL8. Significantly, the Drosophila RPL8 mutant can be rescued by wild-type human RPL8. We also provide genetic evidence that Drosophila RPL8 is a new member of the insulin signaling pathway. In summary, the functions of many human genes appear to be highly conserved, and the ability to identify them in Drosophila represents a powerful genetic tool for large-scale analysis of human transcripts in vivo.
文摘Proteins containing an expanded polyglutamine tract are neurotoxins. The expanded polyglutamine proteins influence a variety of cellular functions. In Drosophila the GMR-Gal4/UAS expression system has been widely used in an eye-based model to study human neurodegenerative diseases. This system has facilitated the isolation and characterization of abundant Drosophilagenes that interact with the expanded polyglutamine proteins. We used the GMR-Gal4/UAS system to express three proteins containing an expanded polyglutamine tract, or an expanded polyglutamine tract alone. Doubling the dose of these proteins resulted in pupal lethality, indicating that these toxic proteins induced a sensitized condition that is prone to synthetic lethality. By using the GMR-Gal4/UAS system, we showed that a Drosophilagene interacts with three expanded polyglutamine proteins to induce a synthetic lethal phenotype. We further demonstrated that the synthetic lethality was mediated through the toxic expanded polyglutamine tract. Our study raises a possibility that conventional genetic screens may not recover synthetic lethal alleles, which are presumably stronger interacting alleles than the currently known modifiers of an expanded polyglutamine tract, due to synthetic lethality.
文摘Mutant proteins containing an expanded polyglutamine tract induce cell death and cause neurodegenerative diseases. These toxic proteins interfere with a variety of physiological pathways, but the key interactions between the toxins and cellular factors remain unclear. To model the diseases in Drosophila, the GMR-Gal4/UAS gene expression system has been used extensively, which operates in the eyes. By using the system, genome-wide studies have resulted in the isolation of functionally diverse groups of Drosophila genes that interact with the disease proteins. We previously reported that coexpressing the Drosophila Dikar gene and an expanded polyglutamine tract by GMR-Gal4/UAS induced a synthetic lethality. We carried out follow-up experiments to isolate additional synthetic lethal alleles. Our data provide evidence that synthetic lethality associated with expressing an expanded polyglutamine tract is more common than thought to be and could have escaped the conventional genetic screens. Our results also suggest that 1) the gene expression system is leaky, allowing expression outside of the primary target eye cell types;2) expressing an expanded polyglutamine tract is extremely toxic to cells;and 3) combining the leaky expression and the toxicity results in a lethal-prone condition. Thus, genetic modifications to the disease proteins’ acute toxicity could frequently lead to synthetic lethality. However, synthetic lethal alleles are excluded from most conventional screens, necessitating alternative approaches such as a two-step method used in this study to isolate the modifiers. Since synthetic lethality reflects essential genetic buffering networks, studying these alleles may hold the keys to identify the critical interactions in the disease development between the toxic proteins and the physiological pathways.
基金Department of Biotechnology,School of Bioengineering,SRM Institute of Science&Technology,Tamil Nadu,India.
文摘Alzheimer’s disease(AD)is one of the most common forms of dementia.Cognitive dysfunction and memory loss are the two main clinical symptoms of AD.Drosophila melanogaster models of AD,which are based on overexpression of human amyloidβ(Aβ)or human tau(hTau)protein,have been used to study the mechanism underlying AD and to screen potential therapeutic compounds.Drugs that are currently available for AD provide only symptomatic relief.Huge unmet medical needs exists to slow,stop,or reverse the progression of AD.Thymoquinone(TQ)is an active ingredient isolated from Nigella sativa(NS)and possesses various pharmacological activities,and it is also a potential neuropharmacological agent.The current study was performed to investigate the effect of dietary administration of TQ at concentrations of 12.5μM and 25μM for 15 and 30 days on biochemical and behavioral parameters,gene,and protein expression of hTau,using Drosophila models of AD.Transgenic Drosophila models exhibiting pan-neuronal and eye-specific expression of hTau were generated using the GAL4/UAS system.Treatment with TQ at both concentrations resulted in a significant increase in behavioral activity,a significant reduction in the amount of reactive oxygen species(ROS),and restoration of depleted superoxide dismutase(SOD)and acetylcholine esterase(AChE)activities.A significant decrease in gene and protein expression of hTau was also observed at the molecular level for both concentrations of TQ.Therefore,TQ has the potential to be investigated as a potential therapeutic phytochemical for the treatment of AD in future studies.
基金This work was supported by the National Natural Science Foundation of China(31801126)the Chongqing Talents:Exceptional Young Talents Project(cstc2022ycjhbgzxm0019)the Doctoral Start-up Foundation of Southwest University(SWU120010).
文摘The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products.In sericulture,only the first filial generation(F1)hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms,but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs.To address this issue,we developed a safe and efficient strategy using the GAL4/Upstream activating sequence(UAS)system,the FLP/flippase recognition target(FRT)system,and the gonad-specific expression gene promoters(RSHP1p and Nanosp)for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms.We established 2 types of activator strains,R1p::GAL4-Gr and Nsp::GAL4-Gr,containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp,respectively,and 1 type of effector strain,UAS::FLP-Rg,containing the UAS-linked FLP gene expression cassette.The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%,whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73%to 80.3%.Additionally,we identified a gene,sw11114,that is expressed in both testis and ovary of Bombyx mori,and can be used to establish novel gonad-specific expression systems in transgenic silkworms.This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.
