加州大学医学院 Teh Ping Lin 博士早在1966年即利用小鼠受精卵原核显微注射技术,系统地研究了微注射不同量外源物质(石蜡油、牛γ-球蛋白溶液)对小鼠胚胎受体移植后的分裂、发育的影响程度,并预见该技术用于研究小鼠或其它种类哺乳动...加州大学医学院 Teh Ping Lin 博士早在1966年即利用小鼠受精卵原核显微注射技术,系统地研究了微注射不同量外源物质(石蜡油、牛γ-球蛋白溶液)对小鼠胚胎受体移植后的分裂、发育的影响程度,并预见该技术用于研究小鼠或其它种类哺乳动物胚胎分化及遗传效应具有潜在价值。直到1980年,随着分子生物学技术的迅速发展,Gordon 等人首先采用显微注射法育成带有人胸苷激酶基因这一外源 DNA 片断的'新型'转基因小鼠后。展开更多
The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell- based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically ...The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell- based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB （OKT3-28BB）. The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co- electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effec- tive method for generating high-quality T cells for adop- tive immunotherapy.展开更多
Objective： To investigate the effects of gene transfer of a β-adrenergic receptor（β-AR） kinase inhibitor（β ARIct） on pulmonary β2-adrenergic receptor and cAMP following β2-AR agonist treatment in asthmatic m...Objective： To investigate the effects of gene transfer of a β-adrenergic receptor（β-AR） kinase inhibitor（β ARIct） on pulmonary β2-adrenergic receptor and cAMP following β2-AR agonist treatment in asthmatic mice, and to analyze the relationship between the routes of gene delivery and the changes of β2AR and cAMP. Methods： BALB/c mice were sensitized and challenged by ovalbumin to establish the asthmatic model treated with βAR agonist （salbutamol injected intramuscularly）. The plasmid with the expression of βARKct was constructed and βARKct gene transfer was performed through intravenous injection or intratracheal instillation in asthmatic mice. The gene expression was measured with Western blot analysis, and the changes of pulmonary β-AR and cAMP evaluated by Radioimmunoassay. Results： The expression of tranfered βARKct gene was detectable in lungs and it was expressed more in the lungs of the mice receiving intratracheally plasmid than those receiving intravenously. The levels of βAR and cAMP were upregulated after using plasmid-βARKct to the asthmatic mice treated with βAR agonist. Conclusion： Our results indicated that there were down-regulation of βAR and cAMP in asthmatic mice treated with βAR agonist. Gene transfer of βARKct could inhibit the extent of the down-regulation of βAR and cAMP. The route of gene delivery could also affect the degree of up-regulation of βAR and cAMP. Gene transfer βARKct may provide a novel approach to the therapeutic strategy for asthma.展开更多
Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to produce m...Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors.We established an efficient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin(LF)gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.展开更多
文摘加州大学医学院 Teh Ping Lin 博士早在1966年即利用小鼠受精卵原核显微注射技术,系统地研究了微注射不同量外源物质(石蜡油、牛γ-球蛋白溶液)对小鼠胚胎受体移植后的分裂、发育的影响程度,并预见该技术用于研究小鼠或其它种类哺乳动物胚胎分化及遗传效应具有潜在价值。直到1980年,随着分子生物学技术的迅速发展,Gordon 等人首先采用显微注射法育成带有人胸苷激酶基因这一外源 DNA 片断的'新型'转基因小鼠后。
文摘The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell- based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB （OKT3-28BB）. The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co- electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effec- tive method for generating high-quality T cells for adop- tive immunotherapy.
文摘Objective： To investigate the effects of gene transfer of a β-adrenergic receptor（β-AR） kinase inhibitor（β ARIct） on pulmonary β2-adrenergic receptor and cAMP following β2-AR agonist treatment in asthmatic mice, and to analyze the relationship between the routes of gene delivery and the changes of β2AR and cAMP. Methods： BALB/c mice were sensitized and challenged by ovalbumin to establish the asthmatic model treated with βAR agonist （salbutamol injected intramuscularly）. The plasmid with the expression of βARKct was constructed and βARKct gene transfer was performed through intravenous injection or intratracheal instillation in asthmatic mice. The gene expression was measured with Western blot analysis, and the changes of pulmonary β-AR and cAMP evaluated by Radioimmunoassay. Results： The expression of tranfered βARKct gene was detectable in lungs and it was expressed more in the lungs of the mice receiving intratracheally plasmid than those receiving intravenously. The levels of βAR and cAMP were upregulated after using plasmid-βARKct to the asthmatic mice treated with βAR agonist. Conclusion： Our results indicated that there were down-regulation of βAR and cAMP in asthmatic mice treated with βAR agonist. Gene transfer of βARKct could inhibit the extent of the down-regulation of βAR and cAMP. The route of gene delivery could also affect the degree of up-regulation of βAR and cAMP. Gene transfer βARKct may provide a novel approach to the therapeutic strategy for asthma.
基金This work was supported by grants from the National High Technology Research and Development Program of China(No.2002AA206621)the Key Programs for Science and Technology Development of Shandong Province(No.J05K06)+2 种基金the Program of Science and Technology Grant of Qingdao City(No.06-2-2-11-jch)Nature Science Foundation of Shandong Province(No.Z2007D01)Doctoral Foundation of Qingdao Agricultural University(No.630615).
文摘Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors.We established an efficient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin(LF)gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.
