绵羊皮肤成纤维细胞对G418及Blasticidin S抗性的研究
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摘要
抗生素常被用于对转基因动物、植物及微生物细胞进行筛选。在进行抗生素抗性筛选前,需要摸索合适的抗生素使用浓度,其原因在于:过低的筛选浓度无法抑制非转基因细胞的生长;反之,过高的筛选浓度会导致转基因细胞死亡。为了摸索绵羊皮肤成纤维细胞(sheep skin fibroblasts, SSFs)对G418及Blasticidin S的抗性,本实验以体外培养的SSFs为实验材料,采用不同浓度的上述两种抗生素处理SSFs,采用活细胞计数的方式检测SSFs对上述两种抗生素的抗性。单独使用G418或Blasticidin S导致SSFs全部死亡的最低致死浓度分别为200μg/mL及4μg/mL;当两种抗生素联合使用时,其最低致死浓度为100μg/mL G418+3μg/mL Blasticidin S或150μg/mL G418+2μg/mL Blasticidin S。该实验为采用这两种抗生素筛选转基因SSFs提供了理论基础。
Antibiotics are often used to screen transgenic animals, plants and microbial cells. Before antibiotic resistance screening, suitable concentration for antibiotics should be explored, which is due to the reason that excessively low screening concentration cannot inhibit the proliferation of non-transgenic cells, on the contrary, e xcessively high screening concentration will lead to the death of transgenic cells. In order to explore the resistance of sheep skin fibroblasts(SSFs) to G418 and Blasticidin S, SSFs cultured in vitro were used as material in this study.SSFs were treated with different concentrations of above two kinds of antibiotics, and the resistance of SSFs to the above two kinds of antibiotics was determined by living cell counting method. The results showed that the minimum lethal concentration of SSFs death when used G418 alone or Blasticidin S alone was 200 μg/mL and 4 μg/mL,respectively. When G418 and Blasticidin S were combined, the minimum lethal concentration was 100 μg/mL G418+3 μg/mL Blasticidin S or 150 μg/mL G418+2 μg/mL Blasticidin S. This study could provise a theoretical basis for the screening of transgenic SSFs using these two kinds of antibiotics.
Antibiotics are often used to screen transgenic animals, plants and microbial cells. Before antibiotic resistance screening, suitable concentration for antibiotics should be explored, which is due to the reason that excessively low screening concentration cannot inhibit the proliferation of non-transgenic cells, on the contrary, e xcessively high screening concentration will lead to the death of transgenic cells. In order to explore the resistance of sheep skin fibroblasts(SSFs) to G418 and Blasticidin S, SSFs cultured in vitro were used as material in this study.SSFs were treated with different concentrations of above two kinds of antibiotics, and the resistance of SSFs to the above two kinds of antibiotics was determined by living cell counting method. The results showed that the minimum lethal concentration of SSFs death when used G418 alone or Blasticidin S alone was 200 μg/mL and 4 μg/mL,respectively. When G418 and Blasticidin S were combined, the minimum lethal concentration was 100 μg/mL G418+3 μg/mL Blasticidin S or 150 μg/mL G418+2 μg/mL Blasticidin S. This study could provise a theoretical basis for the screening of transgenic SSFs using these two kinds of antibiotics.
引文
Chen B.,Shi X.Y.,Xiang N.,and Wang G.L.,2012,Cytoplasm vacuolization of fibroblasts during purification of Schwann cells by geneticin(G418):An optical microscope observation and analysis,Zhongguo Zhuzhi Gongcheng Yanjiu(Journal of Clinical Rehabilitative Tissue Engineering Research),16(14):2593-2596(陈宝,史晓远,向宁,王光林,2012,遗传霉素(G418)纯化许旺细胞过程中成纤维细胞胞浆空泡化的光镜观察,中国组织工程研究,16(14):2593-2596)
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Li Z.M.,2008,Effects of protein Mstl on cell apoptosis and proliferation in HEK293 cell line,Thesis for M.S.,Huazhong University of Science and Technology,Supervisor:Yan Q.,pp.18(李兆明,2008,高表达蛋白Mst1对人胚肾HEK293细胞凋亡和增殖的影响,硕士学位论文,华中科技大学,导师:严群,pp.18)
Ma Y.,Cress W.D.,and Haura E.B.,2003,Flavopiridol-induced apoptosis is mediated through up-regulation of E2F1 and repression of Mcl-1,Mol.Cancer Ther.,2(1):73-81
Sambrook J.,ed.,Huang P.T.,Trans.,2005,Molecular Cloning a laboratory manual(Third Edition),Science Press,Beijing,China,pp.1366-1383(莎姆布鲁克J.,著,黄培堂,译,2005,分子克隆实验指南(第三版),科学出版社,中国,北京,pp.1366-1383)
Wang H.R.,Gu C.H.,Zhong J.H.,Wang T.,Han J.Y.,Chen F.Y.,and Ouyang R.R.,2006,Combined use of flow cytometry and 6418 to screen the recombinant plasmid transfected cells,Zhenduanxue Lilun Yu Shijian(Journal of Diagnostics Concepts and Practice),5(1):52-55(王海嵘,顾春红,钟济华,王婷,韩洁英,陈芳源,欧阳仁荣,2006,流式细胞术结合G418筛选重组质粒稳定转染细胞,诊断学理论与实践,5(1):52-55)
Wang T.,Shi S.B.,and Sha H.Y.,2013,MicroRNAs in regulation of pluripotency and somatic cell reprogramming:small molecule with big impact,RNA Biol.,10(8):1255-1261
Wu J.,2012,Heterologous expression of blasticidin s in S.Lividahs strain and genetic study of the key GENES in the blasticidin S biosynthetic gene cluster,Dissertation for Ph.D.,Shanghai Jiaotong University,Supervisor:Zhou X.F.,pp.90(吴俊,2012,杀稻瘟菌素的异源表达及其基因簇内关键基因的体内功能研究,博士学位论文,上海交通大学,导师:周秀芳,pp.90)
Xiao X.M.,2014,Research on the role of Twist gene in invasion and metastasis of colon cancer cell lines in vitro,Thesis for M.S.,Tianjin Medical University,Supervisor:Liu T.,pp.4-11(肖相明,2014,Twist基因对结肠癌细胞系侵袭转移影响的体外研究,硕士学位论文,天津医科大学,导师:刘彤,pp.4-11)
Zheng Y.M.,Liu F.J.,He X.N.,and Zhang Y.,2006,Study on the hLF gene transgenic goat mammary epithelial cells,Xumu Shouyi Xuebao(Acta Veterinaria Et Zootechnica Sinica),37(12):1282-1286(郑月茂,刘凤军,何小宁,张涌,2006,转人乳铁蛋白基因山羊乳腺上皮细胞的研究,畜牧兽医学报,37(12):1282-1286)
Du A.Q.,Wu J.,Deng Z.X.,and He X.Y.,2014,Determination of the boundary of blasticidin S biosynthetic gene cluster,Weishengwuxue Tongbao(Microbiology China),41(7):1318-1325(杜爱芹,吴俊,邓子新,贺新义,2014,杀稻瘟菌素生物合成基因簇的边界确定,微生物学通报,41(7):1318-1325)
Gruber A.J.,Grandy W.A.,Balwierz P.J.,Dimitrova Y.A.,Pachkov M.,Ciaudo C.,Nimwegen E.,and Zavolan M.,2014,Embryonic stem cell-specific microRNAs contribute to pluripotency by inhibiting regulators of multiple differentiation pathways,Nucleic Acids.Res.,42(14):9313-9326
Li D.,Feng R.C.,Zhang L.X.,Guo F.Q.,Liang Y.,and Teng C.B.,2011,Purification of pancreatic epithelial cells treated with G418 in vitro,Xiandai Shengwu Yixue Jinzhan(Progress in Modern Biomedicine),11(12):2242-2246(李丹,冯锐成,张丽新,郭方琪,梁洋,腾春波,2011,通过G418处理离体纯化小鼠胰腺上皮细胞,现代生物医学进展,11(12):2242-2246)
Li Z.M.,2008,Effects of protein Mstl on cell apoptosis and proliferation in HEK293 cell line,Thesis for M.S.,Huazhong University of Science and Technology,Supervisor:Yan Q.,pp.18(李兆明,2008,高表达蛋白Mst1对人胚肾HEK293细胞凋亡和增殖的影响,硕士学位论文,华中科技大学,导师:严群,pp.18)
Ma Y.,Cress W.D.,and Haura E.B.,2003,Flavopiridol-induced apoptosis is mediated through up-regulation of E2F1 and repression of Mcl-1,Mol.Cancer Ther.,2(1):73-81
Sambrook J.,ed.,Huang P.T.,Trans.,2005,Molecular Cloning a laboratory manual(Third Edition),Science Press,Beijing,China,pp.1366-1383(莎姆布鲁克J.,著,黄培堂,译,2005,分子克隆实验指南(第三版),科学出版社,中国,北京,pp.1366-1383)
Wang H.R.,Gu C.H.,Zhong J.H.,Wang T.,Han J.Y.,Chen F.Y.,and Ouyang R.R.,2006,Combined use of flow cytometry and 6418 to screen the recombinant plasmid transfected cells,Zhenduanxue Lilun Yu Shijian(Journal of Diagnostics Concepts and Practice),5(1):52-55(王海嵘,顾春红,钟济华,王婷,韩洁英,陈芳源,欧阳仁荣,2006,流式细胞术结合G418筛选重组质粒稳定转染细胞,诊断学理论与实践,5(1):52-55)
Wang T.,Shi S.B.,and Sha H.Y.,2013,MicroRNAs in regulation of pluripotency and somatic cell reprogramming:small molecule with big impact,RNA Biol.,10(8):1255-1261
Wu J.,2012,Heterologous expression of blasticidin s in S.Lividahs strain and genetic study of the key GENES in the blasticidin S biosynthetic gene cluster,Dissertation for Ph.D.,Shanghai Jiaotong University,Supervisor:Zhou X.F.,pp.90(吴俊,2012,杀稻瘟菌素的异源表达及其基因簇内关键基因的体内功能研究,博士学位论文,上海交通大学,导师:周秀芳,pp.90)
Xiao X.M.,2014,Research on the role of Twist gene in invasion and metastasis of colon cancer cell lines in vitro,Thesis for M.S.,Tianjin Medical University,Supervisor:Liu T.,pp.4-11(肖相明,2014,Twist基因对结肠癌细胞系侵袭转移影响的体外研究,硕士学位论文,天津医科大学,导师:刘彤,pp.4-11)
Zheng Y.M.,Liu F.J.,He X.N.,and Zhang Y.,2006,Study on the hLF gene transgenic goat mammary epithelial cells,Xumu Shouyi Xuebao(Acta Veterinaria Et Zootechnica Sinica),37(12):1282-1286(郑月茂,刘凤军,何小宁,张涌,2006,转人乳铁蛋白基因山羊乳腺上皮细胞的研究,畜牧兽医学报,37(12):1282-1286)