TITLE: Protocol for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells-Animal
AUTHORS: Marco A. de Oliveira, Lilian H. Florentino, Thais T. Sales, Rayane N. Lima, Luciana R. C. Barros, Cintia G. Limia, Mariana S. M. Almeida, Maria L. Robledo, Leila M. G. Barros, Eduardo O. Melo, Daniela M. Bittencourt, Stevens K. Rehen, Martín H. Bonamino, Elibio Rech

[DESCRIPTION]
This protocol details the assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells in animal.

[STEPS]
SECTION: Cell Passage ● Timing 30 min
1. Cultivate bovine fibroblast cells at 5.0x105 cells per culture flask with a 75 cm2 surface area. Well area coverage, between 60-80% confluence. It takes approximately 8-10 days.
SECTION: Cell Passage ● Timing 30 min
2. Before starting dissociation, place the working aliquot of 0.05% trypsin and EDTA solution in the incubator at 37 °C for at least 10 min.

SECTION: Cell Passage ● Timing 30 min
3. Remove the DMEM culture medium and wash with Dulbecco's Phosphate-Buffered Saline (DPBS, GIBCO). Discard the DPBS and immediately add 2 mL of 0.05% trypsin and EDTA. Make sure to cover the plate area.

SECTION: Cell Passage ● Timing 30 min
4. Place the culture flask in the incubator at 37 °C for 5 min.
 


SECTION: Cell Passage ● Timing 30 min
5. Add 2 mL of DMEM.

SECTION: Cell Passage ● Timing 30 min
6. Transfer the loose cell suspension to a 1.5 or 2 mL centrifugation tube.
SECTION: Cell Passage ● Timing 30 min
7. Spin at 1300 rpm, 5 min.

SECTION: Cell Passage ● Timing 30 min
8. Aspirate and discard the supernatant.
SECTION: Cell Passage ● Timing 30 min
9. Wash with 5 mL of DPBS using a serological pipette.

SECTION: Cell Passage ● Timing 30 min
10. Spin at 1300 rpm, 5 min.

SECTION: Cell Passage ● Timing 30 min
11. Aspirate and discard the supernatant.
SECTION: Cell Passage ● Timing 30 min
12. Resuspend the cell pellet with fresh DMEM medium using a serological pipette.
SECTION: Cell Passage ● Timing 30 min
13. Add 1 mL of cells/flask culture to new culture flasks with a 75 cm2 surface area that have been previously prepared.
 


SECTION: Cell Passage ● Timing 30 min
14. Incubate the cells at 37 °C, 95% humidity, 5% CO2.

SECTION: Cell Passage ● Timing 30 min
15. After 24h, replace the old culture medium with 10 mL of fresh DMEM medium.
 


SECTION: Cell Passage ● Timing 30 min
16. Divide the cells every 5 days.
SECTION: Bovine fibroblast cell transfection with Lipofectamine LTX and Plus Reagent. ● Timing 1h
17. Prepare the cells for transfection: the cells should be at a specific stage of culture and in the appropriate medium for transfection.
SECTION: Bovine fibroblast cell transfection with Lipofectamine LTX and Plus Reagent. ● Timing 1h
18. Prepare the transfection solution: mix the LTX Plus with the desired plasmid following the manufacturer's instructions.
SECTION: Bovine fibroblast cell transfection with Lipofectamine LTX and Plus Reagent. ● Timing 1h
19. Add the transfection solution to the cells: add the transfection solution to cells grown in culture plate or to the medium for suspended cells.
SECTION: Bovine fibroblast cell transfection with Lipofectamine LTX and Plus Reagent. ● Timing 1h
20. Incubate the cells: incubate the cells with the transfection solution for a specific period of time, usually 4-6 hours.
 

SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21. Dissociating cells using 0.05% Trypsin-EDTA (1X):
SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.1. Remove the old growth medium from each well and wash with 0.5 mL/well of DPBS, remove immediately.
SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.2. Add 0.5 mL/well of 0.05% Trypsin-EDTA.

SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.3. Incubate the cells in the incubator at 37 °C for 5 min.
 



SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.4. Add 0.5 mL/well of DMEM in a microcentrifuge and 1.5 or 2 mL tube containing DMEM.

SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.5. Spin at 1300 rpm, 5 min.

SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.6. Wash the cells with DPBS.
SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.7. Spin at 1300 rpm, 5 min.

SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.8. Remove the supernatant and resuspend the cells with DPBS.
SECTION: Analysis of integrase expression in bovine fibroblast cells by flow cytometry. ● Timing 2.5h
21.9. Transfer the cells to a 1.5 mL centrifugation tube for flow cytometry analysis.
 

SECTION: Cell viability assays. ● Timing 7h
22. Count the cells with a Neubauer chamber and cultivate bovine fibroblasts and HEK 293T cells in 96-well plates at a density of 1x105 cells/well in triplicate and grown for 1440 min at 37 °C in a 5% CO2 atmosphere.

SECTION: Cell viability assays. ● Timing 7h
23. After 48 hours of transient cotransfection, incubate the cells with 15 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Thermo Fisher Scientific) (5 mg/mL). 
 


SECTION: Cell viability assays. ● Timing 7h
24. Incubate for 240 min at 37 °C.

SECTION: Cell viability assays. ● Timing 7h
25. Remove the MTT solution and add 150 µL of DMSO to each well to dissolve formazan crystals.
 


SECTION: Cell viability assays. ● Timing 7h
26. Read the absorbance at 595 nm in a plate reader or spectrophotometer.