Two-wavelength fluorescence assay for DNA repair
Two-wavelength fluorescence assay for DNA repair
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DOI：
10.1006/abio.2000.4865
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发表时间：
2000-12-15
影响因子：
2.9
通讯作者：
Russev, G
中科院分区：
文献类型：
--
作者：
Roguev, A;Russev, G
建立了一种简便、可靠的定量测定细胞DNA修复能力的方法。它是基于宿主细胞再活化的紫外线照射质粒pEGFP携带的标记基因的增强型绿色荧光蛋白（EGFP）。作为参考，我们使用携带红移荧光蛋白（EYFP）基因的质粒pEYFP。两种蛋白质都可以被可见光激发，最大值在488 nm，但EGFP在509 nm处发射最大值，而EYFP在527 nm处发射最大值。这使得可以通过测量两个波长的荧光来同时监测两个基因的表达。用紫外线照射的pEGFP和未损伤的pEYFP的混合物共转染HEK293细胞。在转染后的不同时间间隔，相对于EYFP的荧光测定EGFP的荧光，以补偿转染效率或其他实验变量的任何差异。它被用来计算DNA中紫外线损伤的数量，从而计算宿主细胞的修复能力。结果发现，HEK293细胞能够平均在12小时内修复每1000个核苷酸DNA约1.4个UV损伤。(C)北京大学出版社.
A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average. (C) 2000 Academic Press.