【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
Drosophila neurexin(DNRX) plays a critical role in proper architecture development and synaptic function in vivo. However, the temporal and spatial expression pattern of DNRX still remains unclear. For this study, we ...Drosophila neurexin(DNRX) plays a critical role in proper architecture development and synaptic function in vivo. However, the temporal and spatial expression pattern of DNRX still remains unclear. For this study, we generated a novel Drosophila transgenic strain termed the DNRX-Gal4 transgenic line, with characteristic features in agreement with the endogenous DNRX expression pattern. DNRX expression was examined by driving the expression of a GFP reporter(nuclear-localized and membrane-localized GFP) using the DNRX-Gal4 promoter. We found that DNRX was expressed preferentially in central and motor neurons in embryos, larvae and adults, but not in glial cells. DNRX was expressed in pre- and post-synaptic areas in third instar larvae neuromuscular junctions(NMJs). Reporter expression was also observed in the salivary glands, guts, wings and legs of adult flies. In the adult brain, reporter expression was observed throughout several brain regions, including the mushroom body(MBs), antennal lobe(AL) and optic lobe neurons, which is consistent with endogenous DNRX expression via antibody staining. Interestingly, DNRX was also expressed in clock neurons. Meanwhile, we found that DNRX expression in the MBs was required for olfactory learning and memory.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supported by the National Natural Science Founda-tion of China(3117104131000486)the National Basic Research Program of China(2012CB517903)
文摘Drosophila neurexin(DNRX) plays a critical role in proper architecture development and synaptic function in vivo. However, the temporal and spatial expression pattern of DNRX still remains unclear. For this study, we generated a novel Drosophila transgenic strain termed the DNRX-Gal4 transgenic line, with characteristic features in agreement with the endogenous DNRX expression pattern. DNRX expression was examined by driving the expression of a GFP reporter(nuclear-localized and membrane-localized GFP) using the DNRX-Gal4 promoter. We found that DNRX was expressed preferentially in central and motor neurons in embryos, larvae and adults, but not in glial cells. DNRX was expressed in pre- and post-synaptic areas in third instar larvae neuromuscular junctions(NMJs). Reporter expression was also observed in the salivary glands, guts, wings and legs of adult flies. In the adult brain, reporter expression was observed throughout several brain regions, including the mushroom body(MBs), antennal lobe(AL) and optic lobe neurons, which is consistent with endogenous DNRX expression via antibody staining. Interestingly, DNRX was also expressed in clock neurons. Meanwhile, we found that DNRX expression in the MBs was required for olfactory learning and memory.
