目的建立实时定量PCR法检测转基因CHO细胞中外源抗体轻重链基因的拷贝数。方法以分别带有抗体轻链和重链的质粒作为标准品,进行实时定量PCR反应,建立标准曲线。提取转染抗体轻重链基因的CHO细胞基因组DNA,进行定量PCR反应,通过标准曲线...目的建立实时定量PCR法检测转基因CHO细胞中外源抗体轻重链基因的拷贝数。方法以分别带有抗体轻链和重链的质粒作为标准品,进行实时定量PCR反应,建立标准曲线。提取转染抗体轻重链基因的CHO细胞基因组DNA,进行定量PCR反应,通过标准曲线,再根据10 ng CHO基因组中含有的单拷贝基因的数量,分别计算得到抗体的轻链和重链基因在CHO细胞中的拷贝数。结果分别建立了抗体轻链和重链基因的拷贝数标准曲线,标准曲线的相关系数均在0.99以上,PCR扩增效率分别为91.6%和91.8%,具有良好的特异性。随着细胞培养代次的增加,轻链基因和重链基因的拷贝数均出现降低的现象。结论成功建立了实时定量PCR法检测转基因CHO细胞中外源抗体轻链和重链基因的拷贝数,可用于外源抗体基因在CHO细胞中的遗传稳定性研究,也为高表达细胞株的获得提供了一种检测方法。展开更多
The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test cross...The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test crosses, we established an ‘‘all-fish'' growth hormone(GH)transgenic common carp family that exhibits fast growth.In this present study, genome walking, real-time quantitative polymerase chain reaction, and fluorescence in situ hybridization techniques were applied to identify the integration characteristics of the exogenous grass carp GH gene in the transgenic common carp. The exogenous GH genes, in the form of two complete and one incomplete tandem repeats, were found to have integrated into an ATrich region near the end of a chromosome pair. We hypothesize that the high efficiency of exogenous GH gene expression might be due to the low copy number in the genome and the AT-rich integration site.展开更多
Objective To create transgenic mice expressing hamster- and human-PRNP as a model tor understanding the physiological function and pathology of prion protein （PrP）, as well as the mechanism of cross-species transmis...Objective To create transgenic mice expressing hamster- and human-PRNP as a model tor understanding the physiological function and pathology of prion protein （PrP）, as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies （TSEs）. Methods Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin （H ＆ E） staining and immunohistochemical （IHC） methods. Results Integrated PRNP copy number in various mouse lines was 53 （Tg-haPrP1）, 18 （Tg-huPrP1）, 3 （Tg-huPrP2）, and 16 （Tg-huPrP5）, respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs. Conclusion We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.展开更多
文摘目的建立实时定量PCR法检测转基因CHO细胞中外源抗体轻重链基因的拷贝数。方法以分别带有抗体轻链和重链的质粒作为标准品,进行实时定量PCR反应,建立标准曲线。提取转染抗体轻重链基因的CHO细胞基因组DNA,进行定量PCR反应,通过标准曲线,再根据10 ng CHO基因组中含有的单拷贝基因的数量,分别计算得到抗体的轻链和重链基因在CHO细胞中的拷贝数。结果分别建立了抗体轻链和重链基因的拷贝数标准曲线,标准曲线的相关系数均在0.99以上,PCR扩增效率分别为91.6%和91.8%,具有良好的特异性。随着细胞培养代次的增加,轻链基因和重链基因的拷贝数均出现降低的现象。结论成功建立了实时定量PCR法检测转基因CHO细胞中外源抗体轻链和重链基因的拷贝数,可用于外源抗体基因在CHO细胞中的遗传稳定性研究,也为高表达细胞株的获得提供了一种检测方法。
基金supported by the National High Technology Research and Development Program of China(‘‘863’’Program)(2011AA100404,2011AA100403)the National Natural Science Foundation of China(31325026,31272649)
文摘The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test crosses, we established an ‘‘all-fish'' growth hormone(GH)transgenic common carp family that exhibits fast growth.In this present study, genome walking, real-time quantitative polymerase chain reaction, and fluorescence in situ hybridization techniques were applied to identify the integration characteristics of the exogenous grass carp GH gene in the transgenic common carp. The exogenous GH genes, in the form of two complete and one incomplete tandem repeats, were found to have integrated into an ATrich region near the end of a chromosome pair. We hypothesize that the high efficiency of exogenous GH gene expression might be due to the low copy number in the genome and the AT-rich integration site.
基金supported by Chinese National Natural Science Foundation Grants 30771914 and 30800975Institution Technique R&D Grant (2008EG150300)+2 种基金National Basic Research Program of China (973 Program) (2007CB310505)China Mega-Project for Infectious Disease (2009ZX10004-101)the SKLID Development Grant (2008SKLID102 and 2008SKLID202)
文摘Objective To create transgenic mice expressing hamster- and human-PRNP as a model tor understanding the physiological function and pathology of prion protein （PrP）, as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies （TSEs）. Methods Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin （H ＆ E） staining and immunohistochemical （IHC） methods. Results Integrated PRNP copy number in various mouse lines was 53 （Tg-haPrP1）, 18 （Tg-huPrP1）, 3 （Tg-huPrP2）, and 16 （Tg-huPrP5）, respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs. Conclusion We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.
