The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incuba...The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.展开更多
Firefly luciferase (EC1. 13. 12. 7) from Photinus pyralis catalyzes oxydecarboxylation of D-luciferin in the presence of MgATP accompanied by visible light emission with the maximum wavelength of 562 nm. The total rea...Firefly luciferase (EC1. 13. 12. 7) from Photinus pyralis catalyzes oxydecarboxylation of D-luciferin in the presence of MgATP accompanied by visible light emission with the maximum wavelength of 562 nm. The total reaction is as follows:展开更多
文摘The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.
文摘Firefly luciferase (EC1. 13. 12. 7) from Photinus pyralis catalyzes oxydecarboxylation of D-luciferin in the presence of MgATP accompanied by visible light emission with the maximum wavelength of 562 nm. The total reaction is as follows:
文摘目的 构建部分骨钙素启动子控制荧光素酶报告基因的真核表达载体 pc DNA3- OCP- L uc,转染细胞后检测报告基因对 rh BMP- 2的反应 ,以期用于 rh BMP活性的定量测定 .方法 用 PCR方法扩增骨钙素部分启动子 14 7bp,克隆入p GEM- 3zf(- )中 ,经酶切鉴定和序列分析正确后 ,亚克隆入能稳定高效表达荧光素酶的真核表达载体 pc DNA3- L uc中 ,构建真核表达载体 pc DNA3- OCP- L uc,然后分别将 pc DNA3-L uc和 pc DNA3- OCP- L uc转染细胞 ,并检测其对 rh BMP- 2的反应 .结果 成功构建了部分骨钙素启动子控制荧光素酶报告基因的真核表达载体 pc DNA3- OCP- L uc;转染细胞后其报告基因的基础活性较 pc DNA3- L uc大为降低 ,而且报告基因的表达与 rh BMP- 2剂量在一定范围内成线性正相关 .结论 真核表达载体 pc DNA3- OCP- L uc的报告基因表达具有特异性 ,可代替直接测定骨钙素用于 rh BMP- 2活性的定量测定研究 .
