Team:NUDT CHINA/Experiments
Experiments
To create, to dream
Protocol for Agarose Gel Electrophoresis
Protocol for Culture and Subculture
Protocol for Gel Purification with OMEGA Gel Extraction Kit
Protocol for Adenovirus Infection in Mouse Primary Hepatocytes
Protocol for Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit
Protocols of transferrin-lipoplex transfection into mouse primary hepatocytes
Protocol for cell mRNA extraction with Trizol
Protocol for Glucose Concentration Test with Solarbio™ Glucose Detection Kit
Protocol for Glucose Sensing Promoter Test in HepG2 Cell Line
Protocol for Glycogen Test with Solarbio™ Glycogen PAS Dye for Cell
Protocol for Immunoprecipitation with Beyotime™ Protein A+G Agarose
Protocol for lipo3000 transfection with Lipofectamine™ 3000 Reagent
Protocol for Plasmid Extraction with Sangon Biotech™ Sanprep Plasmid Extraction Kit
Protocol for Real-time PCR with NovoStart® SYBR qPCR SuperMix Plus
Material
1. Complete Cell Culture media(400ul/well):Hyclone™ High Glucose Dulbecco's Modified Eagle Medium( DMEM ) + 10% PAN™ Fetal Bovine Serum(FBS) + 1% Hyclone™ 100X Penicillin-Streptomycin Solution(P/S).
2. Glucose-free Cell Culture Media:Gibco™ glucose free Dulbecco's Modified Eagle Medium( DMEM ) +10% PAN™ Fetal Bovine Serum(FBS)+1%Hyclone 100X Penicillin-Streptomycin Solution(P/S).
3. Glucose Stork Solution:1.25mol/L D-Glucose stork solution made of MilliQ™ Deionized Water and Sangon™ Glucose.
4. (Beyotime)Dual Luciferase Reporter Gene Assay Kit
5. Invitrogen LipofectamineTM 3000 Reagent
Method
Figure 1. The simple chart of the experimental step
1. 2 hours before transfection, starve cells using Dulbecco’s Modified Eagle Medium( DMEM ) be cultured in 24-well plate. Before this, it had has 90% HepG2;
2. Dilute 0.75μL lipofectamine3000TM Reagent in 24.25 μL DMEM pre well;
3. Dilute 600 ng DNA and 1ul of P3000 in DMEM and make a total volume of 25 ul per well;
4. Add diluted DNA to diluted LipofectamineTM 3000 Reagent (1:1 ratio);
5. Incubate for 10-15 minutes at room temperature;
6. Change cell culture into complete cell culture medium, add DNA-lipid complex to a well of the 24-well plate;
7. Incubate cells for 12 h at 37°C, 5% CO2, change cell medium into glucose-free complete culture medium.
8. Incubate cells for 6 h at 37°C, 5% CO2, add glucose stock solution and give glucose stimulation.
9. Incubate cells for 6/20 h at 37°C, 5% CO2, collect data by Dual Luciferase Reporter Gene Assay Kit.
Material
1. Complete Cell Culture media(400ul/well): Hyclone™ High Glucose Dulbecco's Modified Eagle Medium( DMEM ) + 10% PAN™ Fetal Bovine Serum(FBS) + 1% Hyclone™ 100X Penicillin-Streptomycin Solution(P/S).
2. Glucose-free Cell Culture Media: Gibco™ glucose free Dulbecco's Modified Eagle Medium( DMEM ) +10% PAN™ Fetal Bovine Serum(FBS)+1%Hyclone 100X Penicillin-Streptomycin Solution(P/S).
3. Glucose Stork Solution: 1.25mol/L D-Glucose stork solution made of MilliQ™ Deionized Water and Sangon™ Glucose.
4. (Beyotime)Luciferase Reporter Gene Assay Kit.
5. Invitrogen LipofectamineTM 3000 Reagent
Method
Figure 2. The simple chart of the experimental step
1. 2 hours before transfection, starve cells using Dulbecco’s Modified Eagle Medium( DMEM ) be cultured in 24-well plate. Before this, it had has 90% HepG2;
Dilute 0.75μL lipofectamine3000TM Reagent in 24.25 μL DMEM pre well;
2. Dilute 600 ng DNA and 1ul of P3000 in DMEM and make a total volume of 25 ul per well;
3. Add diluted DNA to diluted LipofectamineTM 3000 Reagent (1:1 ratio);
4. Incubate for 10-15 minutes at room temperature;
6. Change cell culture into complete cell culture medium, add DNA-lipid complex to a well of the 24-well plate;
7. Incubate cells for 12 h at 37°C, 5% CO2, change cell medium into glucose-free complete culture medium.
8. Incubate cells for 6 h at 37°C, 5% CO2, add glucose stock solution and give glucose stimulation.
9. Incubate cells at 37°C, 5% CO2, collect data of efficiency of transfection via fluorescence microscopy at continous time set after glucose stimulation of 6, 18, 30, 42 and 54 hours.
2019 January
● Brainstorm for 2019 team project. Anchor T2D as the field we want to anchor our project.
● Background information gathering.
2019 February
● Nurse interview and visit in geracomium to gather realistic T2D information and publicity of good lifestyles for the elders to avoid or retard T2D complication.
● Determine 2019 project --- continue with PR Predator in 2018, but focus on the degradation of glucagon receptor in hepatocytes.
● Look up for authority articles.
● Experimental skills and safety training for team members in Wet Lab.
2019 March
● Crucial parts construction.
2019 April
● First milestone in project --- designed gene circuit works well in degrade GCGR in HepG2 cell line.
2019 June
● Glucose sensing promoter related circuit construction.
2019 July
● Glucose sensing promoter functional test in HepG2.
2019 August
● Result for glucose sensing promoter functional test.
●Determination: Mouse primary hepatocytes as platform for continuous experiments of designed GCGR degradation circuit. Adenovirus as circuit transport tool.
2019 September
●Third milestone: Result for glucose sensing enabled GCGR degradation circuit in HepG2.
2019 October
● Fourth milestone: Result for mouse primary hepatocytes.
●Fifth milestone: Model for evaluate the effectiveness of initial design and indicate the potentials of circuit in long-term in vivo uses.