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Characterization: To characterize the PGK promoter at different time, we used firefly luciferase as its reporter gene. For method, we transfected equal quantity of pmirGLO into liver carcinoma cell line HepG2. Cells were cultured in High Glucose Dulbecco's Modified Eagle Medium with 10% FBS and under condition of 37℃, 5% CO2. 0, 6, 12 and 24h after transfection, cells were dissociated for luciferases (both firefly and renilla). Data was collected by Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit and analyzed by ImageJ. Results are shown below. | Characterization: To characterize the PGK promoter at different time, we used firefly luciferase as its reporter gene. For method, we transfected equal quantity of pmirGLO into liver carcinoma cell line HepG2. Cells were cultured in High Glucose Dulbecco's Modified Eagle Medium with 10% FBS and under condition of 37℃, 5% CO2. 0, 6, 12 and 24h after transfection, cells were dissociated for luciferases (both firefly and renilla). Data was collected by Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit and analyzed by ImageJ. Results are shown below. | ||
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Revision as of 07:04, 21 October 2019
PGK, mammalian promoter
Promoter for expression of genes in mammalian cells
Contribution: NUDT_CHINA 2019
Summary：in this part, we made contributions to the quantitative characterization data of mammalian PGK Promoter. We measured the strength of this promoter by expression change of firefly luciferase at different time.
Characterization: To characterize the PGK promoter at different time, we used firefly luciferase as its reporter gene. For method, we transfected equal quantity of pmirGLO into liver carcinoma cell line HepG2. Cells were cultured in High Glucose Dulbecco's Modified Eagle Medium with 10% FBS and under condition of 37℃, 5% CO2. 0, 6, 12 and 24h after transfection, cells were dissociated for luciferases (both firefly and renilla). Data was collected by Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit and analyzed by ImageJ. Results are shown below.
Figure 1. PGK-drived firefly luciferase RLU in HepG2 cell line after transfection and culturing for 0, 6, 12 and 24 hours.
Conclusions drawn from the results that within 24h, initiation strength of the mammalian PGK promoter keeps rising and reached maximum at 24h after transfection. Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 143
Illegal PstI site found at 400 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 143
Illegal PstI site found at 400 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 143
Illegal PstI site found at 400 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 143
Illegal PstI site found at 400 - 1000COMPATIBLE WITH RFC[1000]