AIM To compare the effects of liposomes andglyco-poly-L-lysine on liver targeted uptake andexpression of plasmid in rat liver.METHODS After binding with lipofectamine orgalactose-terminal glyco-poly-L-lysine,theplasmi...AIM To compare the effects of liposomes andglyco-poly-L-lysine on liver targeted uptake andexpression of plasmid in rat liver.METHODS After binding with lipofectamine orgalactose-terminal glyco-poly-L-lysine,theplasmid could be expressed in eukaryotic cellswhen injected into Wistar rats by intravenousroute.At different time intervals after the injection,the distribution and expression of the plasmid inliver of rats were observed and compared using insitu hybridization and immunohistochemistry.RESULTS The expression of the plasmid bindingto liposomes or G-PLL could be markedly observed24 h later,and began to decrease one week later,but it still could be observed up to three weeks.Both liposomes and G-PLL could deliver theplasmid to the liver effectively,but the effect of thelatter was better than the former concerning thedistribution and expression of the plasmid targeteduptake in the liver.CONCLUSION G-PLL is better than liposome asthe targeted carrier for delivering exogenous genesto the liver.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
An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, t...An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chickswere raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female trans-genic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination ac-cording to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the em-bryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As aresult, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts fromembryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of in-jected MiwZ in the extracts from sperm was also confirmed. The exogenous gene introduced into the GCR migrated suc-cessfully to the gonad resulting in its incorporation into the offspring and spermatozoa of transgenic chickens. (Asian JAndrol 1999 Sep ; 1: 139 - 144)展开更多
Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread ...Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.展开更多
Objectives] This study was conducted to investigate the regulatory mechanism of ethylene on DpXTH gene expression in dahlia ( Dahlia pinnata Cav.) flower petals.[Methods] Dahlia flower petals were treated with ethep...Objectives] This study was conducted to investigate the regulatory mechanism of ethylene on DpXTH gene expression in dahlia ( Dahlia pinnata Cav.) flower petals.[Methods] Dahlia flower petals were treated with ethephon (ETH) and 1-methylcyclopropene (1-MCP) at different flower development stages and for different durations in vivo . Then, the relative expression of DpXTH1 and DpXTH2 in flower samples in different treatments was quantified with real-time PCR, using β-actin as a reference gene.[Results] The expression of DpXTH1 and DpXTH2 in ETH- or 1-MCP-treated dahlia flower petals increased at first, and decreased subsequently with increasing duration of treatment. The relative expression of both DpXTH1 and DpXTH2 in ETH-treated samples reached the maximum level at 12 h. Moreover, the relative expression of both DpXTH1 and DpXTH2 in dahlia flower petals treated with ETH at early stage of flowering was higher than that of the samples treated at other flowering stages; in 1-MCP-treated samples, it was higher at full-flowering stage than at other stages. The maximum relative expression levels of DpXTH1 and DpXTH2 in ETH- and 1-MCP-treated samples were all higher than those of control.[Conclusions] Exogenous ethylene can accelerate the opening of dahlia flowers, and 1-MCP can inhibit this process, so they can be used to regulate development of dahlia flowers.展开更多
To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly ...To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.展开更多
Objective: To explore the possible biological function of human nuclear receptor hLRH-1 in tumorigenesis and progress of colon cancer. Methods: Plasmids pcDNA3-hLRH-1 were introduced into SW480 cells via lipofectami...Objective: To explore the possible biological function of human nuclear receptor hLRH-1 in tumorigenesis and progress of colon cancer. Methods: Plasmids pcDNA3-hLRH-1 were introduced into SW480 cells via lipofectamine. The expression of mRNA and protein of exogenous hLRH-1 were detected by RT-PCR and western blotting, respectively. MTT assay was carried out to survey the proliferation of SW480 cells with overexpression of hLRH-1. Meanwhile, the expression of proliferation-related genes cyclin E1 and cyclin D1, and apoptosis-related genes PTEN and Rbl, were analyzed by realtime RT-PCR. Results: The proliferation of SW480 cells was promoted under the condition of overexpression of hLRH-1. The expression of cyclin E1 was up-regulated significantly, while that of PTEN and Rbl were down-regulated in SW480 cells with overexpressed hLRH-1. Conclusion: The expression of exogenous hLRH-1 in SW480 cells induced the proliferation resulting form up-regulation of cyclin E1, as well as participated in the regulation of apoptosis via influencing the expression of PTEN and Rb1.展开更多
Gene transfer methods are developing quickly recently, but each method has its limitations. We introduce a new gene transfer technique in this paper, which is simple, effective, and easy to operate,but does not get en...Gene transfer methods are developing quickly recently, but each method has its limitations. We introduce a new gene transfer technique in this paper, which is simple, effective, and easy to operate,but does not get enough attention from scientists. This technique is used to transform plants by in jecting exogenous DNA to stigma, style, ovary, young fruit or meristem of the recipient, or soaking the recipient's seeds in exogenous DNA solution. Lots of heritable variations were found in many characters of many crops. It may be used to create new germplasms or realize gene exchange between different species, genera, or families, even between animals and plants. A brief discussion was given to the mechanism of exogenous DNA introduction, integration into and expression in the recipient. We also discussed the merits and limitations of the technique.Currently there are two successful approaches that can be used to transform paints genetically,but each method has its limitations that are delaying the application of the techniques to certain commercially important crops. The first technique exploits a natural genetic engineer, Agrobacterium tumefaciens, which contains a tumor-inducing (Ti) plasmid that transfers a DNA segment (the T-DNA) from the plasmid to the nuclear genome of infected plants (or in vitro to plant tissue). The method is restricted to dicotyledenous plants; monocotyledenous plants are usually not susceptible to agrobacterial infection. The second technique involves direct transfer of DNA to plant protoplast, prepared by enzymatic digestion of cell walls, for example by chemically stimulated uptake using polyethylene glycol or a high voltage pulse, generating transient 'holes' in the protoplast membrane. This technique depends on a tissue culture system that allows regeneration of mature plants from protoplasts. But so far it is impossible to achieve plant regeneration from protoplasts in many crops. Both techniques use dominant selectable markers (for example, kanamycin resistance) to select for the transformed tissue or plant which can then be screened for expression of co-transferred but unselected genes (Lichenstein, 1987).Now there is a new successful method which can transform various crops, regardless of dicots or monocots, cereals or legumes. It doesn't need Agrobacterium tumefaciens and plasmid, doesn't depend on the tissue culture system that allows regeneration of mature plants from protoplasts.Comple and advance equipments are not necessary. It is very simple, but very effective. Next is a review about the technique, its application in several crops, the mechanism of transformation, and its merits and limitations.展开更多
[Objective] This study aimed to investigate the functions and related mechanisms of xyloglucan Endotransglycosylase/hydrolases (XTHs) during the growth and development of dahlia. [Method] Using /3-actin as the refer...[Objective] This study aimed to investigate the functions and related mechanisms of xyloglucan Endotransglycosylase/hydrolases (XTHs) during the growth and development of dahlia. [Method] Using /3-actin as the reference gene, the rela- tive transcription levels of DpXTH1 and DpXTH2 genes in roots, stems, leaves and petals of dahlia were analyzed by real-time RT-PCR. [Result] The DpXTH1 and DpXTH2 were not expressed in the roots, but expressed abundantly in the petals of dahlia. There were little expressions in the stems and leaves of dahlia. [Conclusion] The DpXTH1 and DpXTH2 were petal-specific genes and closely related to the growth and development of petals in dahlia.展开更多
Preliminary basic research and clinical findings have demonstrated that electroacupuncture ther- apy exhibits positive effects in ameliorating depression. However, most studies of the underlying mechanism are at the s...Preliminary basic research and clinical findings have demonstrated that electroacupuncture ther- apy exhibits positive effects in ameliorating depression. However, most studies of the underlying mechanism are at the single gene level; there are few reports regarding the mechanism at the whole-genome level. Using a rat genomic gene-chip, we profiled hippocampal gene expression changes in rats after electroacupuncture therapy. Electroacupuncture therapy alleviated depres- sion-related manifestations in the model rats. Using gene-chip analysis, we demonstrated that electroacupuncture at Baihui (DU20) and Yintang (EX-HN3) regulates the expression of 21 genes. Real-time PCR showed that the genes Vgf, lgf2, Trnp32, Loc500373, Hifla, Folrl, Nrnb, and Rtn were upregulated or downregulated in depression and that their expression tended to nor- malize after electroacupuncture therapy. These results indicate that electroacupuncture at Baihui and Yintang modulates depression by regulating the expression of particular genes.展开更多
基金the National Natural Science Foundation of China(№39570336).
文摘AIM To compare the effects of liposomes andglyco-poly-L-lysine on liver targeted uptake andexpression of plasmid in rat liver.METHODS After binding with lipofectamine orgalactose-terminal glyco-poly-L-lysine,theplasmid could be expressed in eukaryotic cellswhen injected into Wistar rats by intravenousroute.At different time intervals after the injection,the distribution and expression of the plasmid inliver of rats were observed and compared using insitu hybridization and immunohistochemistry.RESULTS The expression of the plasmid bindingto liposomes or G-PLL could be markedly observed24 h later,and began to decrease one week later,but it still could be observed up to three weeks.Both liposomes and G-PLL could deliver theplasmid to the liver effectively,but the effect of thelatter was better than the former concerning thedistribution and expression of the plasmid targeteduptake in the liver.CONCLUSION G-PLL is better than liposome asthe targeted carrier for delivering exogenous genesto the liver.
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.
文摘An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chickswere raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female trans-genic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination ac-cording to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the em-bryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As aresult, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts fromembryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of in-jected MiwZ in the extracts from sperm was also confirmed. The exogenous gene introduced into the GCR migrated suc-cessfully to the gonad resulting in its incorporation into the offspring and spermatozoa of transgenic chickens. (Asian JAndrol 1999 Sep ; 1: 139 - 144)
基金This project was supported by the Washington State University Start-up Funds, George W. Bagby Research Fund
文摘Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.
基金Supported by Applied Basic Research Project of Suzhou City(SYN201405)
文摘Objectives] This study was conducted to investigate the regulatory mechanism of ethylene on DpXTH gene expression in dahlia ( Dahlia pinnata Cav.) flower petals.[Methods] Dahlia flower petals were treated with ethephon (ETH) and 1-methylcyclopropene (1-MCP) at different flower development stages and for different durations in vivo . Then, the relative expression of DpXTH1 and DpXTH2 in flower samples in different treatments was quantified with real-time PCR, using β-actin as a reference gene.[Results] The expression of DpXTH1 and DpXTH2 in ETH- or 1-MCP-treated dahlia flower petals increased at first, and decreased subsequently with increasing duration of treatment. The relative expression of both DpXTH1 and DpXTH2 in ETH-treated samples reached the maximum level at 12 h. Moreover, the relative expression of both DpXTH1 and DpXTH2 in dahlia flower petals treated with ETH at early stage of flowering was higher than that of the samples treated at other flowering stages; in 1-MCP-treated samples, it was higher at full-flowering stage than at other stages. The maximum relative expression levels of DpXTH1 and DpXTH2 in ETH- and 1-MCP-treated samples were all higher than those of control.[Conclusions] Exogenous ethylene can accelerate the opening of dahlia flowers, and 1-MCP can inhibit this process, so they can be used to regulate development of dahlia flowers.
文摘To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.
基金the Young Scientific and Technical Innovation Foundation of Fujian Province (No. 2004J067)Foundation of Fuzhou General Hospital (No. 200638)
文摘Objective: To explore the possible biological function of human nuclear receptor hLRH-1 in tumorigenesis and progress of colon cancer. Methods: Plasmids pcDNA3-hLRH-1 were introduced into SW480 cells via lipofectamine. The expression of mRNA and protein of exogenous hLRH-1 were detected by RT-PCR and western blotting, respectively. MTT assay was carried out to survey the proliferation of SW480 cells with overexpression of hLRH-1. Meanwhile, the expression of proliferation-related genes cyclin E1 and cyclin D1, and apoptosis-related genes PTEN and Rbl, were analyzed by realtime RT-PCR. Results: The proliferation of SW480 cells was promoted under the condition of overexpression of hLRH-1. The expression of cyclin E1 was up-regulated significantly, while that of PTEN and Rbl were down-regulated in SW480 cells with overexpressed hLRH-1. Conclusion: The expression of exogenous hLRH-1 in SW480 cells induced the proliferation resulting form up-regulation of cyclin E1, as well as participated in the regulation of apoptosis via influencing the expression of PTEN and Rb1.
文摘Gene transfer methods are developing quickly recently, but each method has its limitations. We introduce a new gene transfer technique in this paper, which is simple, effective, and easy to operate,but does not get enough attention from scientists. This technique is used to transform plants by in jecting exogenous DNA to stigma, style, ovary, young fruit or meristem of the recipient, or soaking the recipient's seeds in exogenous DNA solution. Lots of heritable variations were found in many characters of many crops. It may be used to create new germplasms or realize gene exchange between different species, genera, or families, even between animals and plants. A brief discussion was given to the mechanism of exogenous DNA introduction, integration into and expression in the recipient. We also discussed the merits and limitations of the technique.Currently there are two successful approaches that can be used to transform paints genetically,but each method has its limitations that are delaying the application of the techniques to certain commercially important crops. The first technique exploits a natural genetic engineer, Agrobacterium tumefaciens, which contains a tumor-inducing (Ti) plasmid that transfers a DNA segment (the T-DNA) from the plasmid to the nuclear genome of infected plants (or in vitro to plant tissue). The method is restricted to dicotyledenous plants; monocotyledenous plants are usually not susceptible to agrobacterial infection. The second technique involves direct transfer of DNA to plant protoplast, prepared by enzymatic digestion of cell walls, for example by chemically stimulated uptake using polyethylene glycol or a high voltage pulse, generating transient 'holes' in the protoplast membrane. This technique depends on a tissue culture system that allows regeneration of mature plants from protoplasts. But so far it is impossible to achieve plant regeneration from protoplasts in many crops. Both techniques use dominant selectable markers (for example, kanamycin resistance) to select for the transformed tissue or plant which can then be screened for expression of co-transferred but unselected genes (Lichenstein, 1987).Now there is a new successful method which can transform various crops, regardless of dicots or monocots, cereals or legumes. It doesn't need Agrobacterium tumefaciens and plasmid, doesn't depend on the tissue culture system that allows regeneration of mature plants from protoplasts.Comple and advance equipments are not necessary. It is very simple, but very effective. Next is a review about the technique, its application in several crops, the mechanism of transformation, and its merits and limitations.
基金Supported by Applied Basic Research Project of Suzhou City(SYN201405)National College Students’Innovative Entrepreneurial Training Program(201410285049Z)~~
文摘[Objective] This study aimed to investigate the functions and related mechanisms of xyloglucan Endotransglycosylase/hydrolases (XTHs) during the growth and development of dahlia. [Method] Using /3-actin as the reference gene, the rela- tive transcription levels of DpXTH1 and DpXTH2 genes in roots, stems, leaves and petals of dahlia were analyzed by real-time RT-PCR. [Result] The DpXTH1 and DpXTH2 were not expressed in the roots, but expressed abundantly in the petals of dahlia. There were little expressions in the stems and leaves of dahlia. [Conclusion] The DpXTH1 and DpXTH2 were petal-specific genes and closely related to the growth and development of petals in dahlia.
基金supported by the National Natural Science Foundation of China,No.81273847
文摘Preliminary basic research and clinical findings have demonstrated that electroacupuncture ther- apy exhibits positive effects in ameliorating depression. However, most studies of the underlying mechanism are at the single gene level; there are few reports regarding the mechanism at the whole-genome level. Using a rat genomic gene-chip, we profiled hippocampal gene expression changes in rats after electroacupuncture therapy. Electroacupuncture therapy alleviated depres- sion-related manifestations in the model rats. Using gene-chip analysis, we demonstrated that electroacupuncture at Baihui (DU20) and Yintang (EX-HN3) regulates the expression of 21 genes. Real-time PCR showed that the genes Vgf, lgf2, Trnp32, Loc500373, Hifla, Folrl, Nrnb, and Rtn were upregulated or downregulated in depression and that their expression tended to nor- malize after electroacupuncture therapy. These results indicate that electroacupuncture at Baihui and Yintang modulates depression by regulating the expression of particular genes.