Team:NUDT CHINA/Contribution

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Contribution

Always looking for better

Archives

• SV40
• PGK
• EF1α

Characterization for exist part

SV40 promoter

    It is a promoter gene derived by the SV40. At pmirGLO Vector, SV40 early enhancer/promotor is located at 426–844 nucleotides, with a length of 419 nucleotides. It initiates the transcription of the hRluc-neo fusion protein coding region. To character its experimental data, we do an experiment about the strength of SV40 promoter at different time.

    In our experiment, we transferred the pmirGLO plasmid into the HepG2 via Lipofectamine™3000. To quantify the reliability of SV40 promoter characteristics with time, we made statistics of its downstream Renilla Luciferase expression as representation of SV40 activity. After transfection of 0h, 6h, 12h, 24h, we gathered cells to detect the expression of hRluc， collected data by Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit and analyzed them by ImageJ. Click BBa_K2440005 for experiment data.

Figure 1. Schematic representation of experiment design. hRluc was used as the reporter gene for SV40.

PGK promoter

    It is a promoter for expression of genes in mammalian cells. To character its experimental data, we do an experiment about the activity of PGK promoter at different time.

    In our experiment, we transferred 500ng pmirGLO Vector into each well of 24 well plate of HepG2. Cells were cultured in High Glucose Dulbecco's Modified Eagle Medium with 10% FBS and under condition of 37℃, 5% CO2. Because the Rluc hasn’t 100% expression rate, so we made statistic of the rate of Renilla Luciferase expression and Firefly lucifferase expression as represent of the PGK activity at different time. We used the Dual Luciferase Reporter Gene Assay Kit and to collect data and ImageJ to analyze data. Click BBa_K2110012 for experiment data.

Figure 2. Schematic representation of experiment design. hRluc and Rluc was used as the reporter gene for PGK.

Human Elongation Factor-1alpha promoter

    This promoter regulates gene expression in mammalian cells. To character its experimental data, we do an experiment about the reliability of EF1α promoter at different time.

    In our experiment, pcDNA3.1-9XGSP-GFP was cotransfected with plv-mCherry by Lipofectamine™3000 into HepG2. We measured the strength of this promoter by expression change of mCherry at continous time set after transfection of 6, 18, 30 and 54 hours. Click BBa_K1119010 for experiment data.

Figure 3. Schematic representation of experiment design. mCherry was used as the reporter gene for EF1α.