UTILITY OF FIREFLY LUCIFERASE AS A REPORTER GENE FOR PROMOTER ACTIVITY IN TRANSGENIC MICE
UTILITY OF FIREFLY LUCIFERASE AS A REPORTER GENE FOR PROMOTER ACTIVITY IN TRANSGENIC MICE
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DOI：
10.1093/nar/16.9.4159
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发表时间：
1988-05-11
影响因子：
14.9
通讯作者：
SMITH, RG
中科院分区：
文献类型：
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作者：
DILELLA, AG;HOPE, DA;SMITH, RG
萤火虫（Photinus pnralis）（萤火虫）荧光素酶测定是极其快速、廉价、非常灵敏的，并且利用市售的非放射性底物。在CV-1细胞中监测真核启动子活性时，荧光素酶测定比氯霉素乙酰转移酶表达测定更灵敏（1）。我们报告的萤火虫荧光素酶基因的效用，以监测转基因小鼠的启动子活性。我们克隆了鸡α-骨骼肌动蛋白启动子的一个小DNA片段下游的荧光素酶基因。该片段包括转录起始位点和200个碱基对的5 '-侧翼序列（2）。杂交基因用于产生表达荧光素酶基因产物的转基因小鼠。通过利用小鼠尾部含有骨骼肌的事实，可以使用尾部活检样品非侵入性地测量骨骼肌肌动蛋白启动子的荧光素酶表达。从73只4天大的FO转基因后代中采集尾部活组织检查。样品制备和荧光素酶测量如（1）所述。表1显示在10只（14%）新生儿中检测到荧光素酶活性，动物可分为低、中和高荧光素酶表达者。通过DNA斑点印迹分析证明这些动物中转基因的存在（数据未显示）。在对照动物中未检测到荧光素酶活性。
Photinus pnralis (firefly) luciferase assays are extremely rapid, inexpensive, very sensitive and utilize commercially available non-radioactive substrates. The luciferase assay is more sensitive than assaying chloramphenicol acetyltransferase expression when monitoring eukaryotic promoter activity in CV-1 cells (1). We report the utility of the firefly luciferase gene to monitor promoter activity in transgenic mice. We cloned the luciferase gene downstream of a small DNA fragment of the chicken alpha-skeletal actin promoter. This fragment included the transcription start site and 200 base pairs of 5'-flanking sequences (2). The hybrid gene was used to generate transgenic mice expressing the luciferase gene product. By exploiting the fact that the mouse tail contains skeletal muscle, expression of luciferase by the skeletal muscle actin promoter could be measured non-invasively using tail biopsy samples. Tail biopsies were taken from 73 4-day-old FO-transgenic offspring. Sample preparations and luciferase measurements were as described (1). Table 1 shows that luciferase activitywas detected in 10 (14%) of the neonates, and that animals could be classified as low, medium, and high luciferase expressors. The presence of the transgene in these animals was demonstrated by DNA dot-blot analysis (data not shown). Luciferase activity was not detected in control animals.