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HepG2 Luciferase Expression Assay with Glutamine Treatment

This study investigates the effect of glutamine treatment on luciferase expression in HepG2 cells transfected with various plasmids. Results show variability in expression levels across different constructs and cell lines.

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HepG2 Luciferase Expression Assay with Glutamine Treatment

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  1. We have some Chr2 strains frozen back but Angie has the information.

  2. A,B,&D strains we have frozen sperm. Angie has frozen down some embryos but I don’t which strains.

  3. HepG2 Transfection ~20%

  4. HepG2 Retrovirus Infection ~50-60%

  5. HepG2 Retroviral Infection No filter

  6. HepG2 Retrovirus Infection Brightfield Image

  7. Aml-12 GFP Retrovirus ~90% infection

  8. Aml-12 Brightfield Image

  9. Pepck Project • Transformed and grew up stocks of plasmids. • Designed primers and sequence verified Pepck plasmids. • pp132 contains -468 to +69 of Pepck Promotor • p490 contains -490 to +70 of Pepck Promotor • p2000 contains ~2300 bp of Pepck Promotor • Exp#1 -Transfected Huh6 with plasmid and treated with and without L glut. • Sequence verified Pepck plasmids

  10. Experiment #1- with Lisa • Reverse transfected Huh6 cells with plasmids and treated with and without L glut. • Performed the Dual-Luciferase Assay the next day. • Results: Showed that the addition of glutamine repressed the expression of the luciferase expression. The opposite result as we expected. • Repeated the experiment, with the same results.

  11. Experiment#3 • Reverse transfected HepG2 cells with plasmids and treated with and without L glut. • Performed the Dual-Luciferase Assay the next day. • Results: Showed a nice increase in luciferase expression but the baseline was high. Had a 2 fold induction in p132 and p2000. p490 showed no change.

  12. Experiment#4 • Reverse transfected HepG2 cells with plasmids and treated with and without L glut. • Performed the Dual-Luciferase Assay the next day. • Results: Showed a 2 fold induction of luciferase expression in p490, but p132 and p2000 showed no change. Lots of variability.

  13. Experiment#5 • Reverse transfected Aml-12 cells with plasmids and treated with and without L glut. • Performed the Dual-Luciferase Assay the next day. • Results: Showed a very low transfection rate, had a 1.5 fold increase in p132 and p490, but nothing in p2000. Stopped this cell line due to the low transfection rate.

  14. Experiment#6 • Reverse transfected HepG2 cells with plasmids and treated with and without L glut. • Performed the Dual-Luciferase Assay the next day. • Results: Showed all three constructs have ~1.6 fold increase in luciferase expression but still have a high baseline.

  15. Experiment#7-Donnie only • Reverse transfected HepG2 cells with plasmids and treated with and without L glut. • Performed the Dual-Luciferase Assay the next day. • Results: Showed all three constructs have ~1.4 to 2 fold increase in luciferase expression but still have a high baseline but the variability went down.

  16. Experiment#8-10Nicole & Donnie • RT, No Trans, and Trans after overnight plating. Then treat plasmids with and without L glut for 24 hours. • Performed the Dual-Luciferase Assay the next day. • Results: The overnight plating then transfection showed the best results. Cells need to be happy to show glutamine effect.

  17. Stopping Point • Next experiment was to be testing retrovirus, lipofectamine, and no treatment on the response of the cells to glutamine. • The hope is retrovirus will be less harsh on the cells than lipofectamine and we will get a better response to glutamine. • If true, then we will have to clone our constructs into retro or lentivirus.

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