Protocol for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells-Human

After performing the sterilization procedure in the safety cabinet, couple the syringe containing 20mL PBS to the leukocyte filter, cut the filter tube extremity, and put it into a 50 mL tube.

Press the syringe to wash the leukocyte filter. Repeat this step if more cells are needed.

With a 25 mL serological pipette, add ~35mL of blood at the top of the 10 mL tube containing Ficoll-Paque at Room temperature.

Centrifuge at 800x g, in a swinging-bucket rotor, without breaking.

With a 10 mL serological pipette, very carefully remove the PBMC “white ring” on top of the Ficoll-Paque and put it into a fresh tube.

To wash the cells, complete the volume to 50mL with PBS and centrifuge at 400x g,10°C. Remove the supernatant.

Repeat the wash step twice.

Resuspend the cells in 5mL-10mL PBS and maintain them On ice.

Count the cells with a Neubauer chamber by adding Trypan blue solution at 4% for cell viability staining.

Add 10x10⁶ cells per electroporation reaction separating in individual microtubes.

Centrifuge the tubes at 400x g,10°C.

Remove all supernatant with a pipette and resuspend the cells in a total of 100µL of 1SM electroporation buffer + plasmids (integrase + target) per reaction.

Immediately insert the cells in the cuvette and electroporate using program U-014 on Nucleofector 2b.

Very carefully, resuspend the cells in warm RPMI/FBS 20% (no antibiotics) and plate them in total 500µL in a 12-well plate. After 16 hours, add 500µL RPMI/FBS 10% + P/S antibiotics + 50 U/mL IL2 + L-Glutamine.

Collect the cells and place them in a microtube.

Centrifuge at 400x g. Remove the supernatant.

Wash the cells with 1mL of cold PBS. If DNA will be extracted, separate an aliquot at this time.

Repeat the centrifugation 2X to wash the cells.

Resuspend the cells in 300µL of cold PBS and place them in the flow cytometer tub.

Add 5µL of 7-AAD and incubate at 4°C for 0h 20m 0s.

Acquire at least 10,000 cells at the viable gate to evaluate eGFP expression. For a higher sensitivity, acquire more cells.

Plate 4x10⁶ HEK293T cells per well of a 75 cm² plate at least 16 h before transfection in DMEM complete medium.

Replace the medium before transfection, adding only 10mL of DMEM complete medium.

Mix the plasmids (integrase + targets, 5µg each) to 500µL of 2X CaCl₂.

Next, agitate CaCl₂ using a vortex at full speed and add 500µL of the HBS solution drop-by-drop very slowly. Make bubbles on the solution with a Pasteur pipette. Let it rest for an instant.

Very carefully add drop-by-drop at the cells covering all the flask with circular movements avoiding two drops at the same spot. Mix the solution with ∞ movement and place the cells in the incubator.

Integrase activity was evaluated by flow cytometry 24 h after transfection. For optimization, several time points should be evaluated. In our hands, 72 h after electroporation, eGFP expression achieved its maximum (1-35% of positive cells).

Culture hES cells in a 100-mm culture dish until the cells cover the dish area at 60-70% confluence. This occurs at approximately 5-6 days.

Prior to starting the dissociation, coat the fresh 100-mm culture dishes with 6 mL/dish of cold Geltrex™ and place it into the incubator at 37°C for at least 0h 30m 0s.

Remove Geltrex™, and immediately add 9 mL/dish of fresh growth mTeSR™ 1 medium to prevent drying.

Place the fresh culture dishes into the incubator at 37°C.

Carefully aspirate the old growth medium from the cell culture dish.

Wash the dish with 4mL of DPBS without calcium and magnesium (DPBS-/-) and remove immediately.

Add 4mL of Accutase™ into the 100-mm culture dish, ensure that cover of dish area.

Incubate the cell suspension at 37°C for 0h 5m 0s.

Add 3mL of DMEM/F12 and gently triturate the clusters across the dish surface.

Scrape the attached cells using a cell scraper.

Transfer the unattached cell suspension into a 15 mL conical tube containing 3mL of DMEM/F12 using a 5 mL serological pipette.

Spin down at 100x g,25°C.

Aspirate and discard the supernatant.

Resuspend the cell pellet with 5mL of fresh growth mTeSR™ 1 Medium using a 5 mL serological pipette.

Add the cell suspension 1 mL/dish into fresh 100-mm culture dishes prepared previously at step 28-31.

Incubate the cells at 37°C, 95% O2, 5% CO₂ in humidified air.

After 24 h, replace the old growth medium with fresh mTeSR™ 1 medium.

Split the cells every 5-6 days.

Carefully aspirate the old growth medium from the cell culture dish.

Replace with 10mL of fresh growth mTeSR™ 1 Medium.

Incubate the cells at 37°C, 95% O2, and 5% CO₂ in humidified air until 60-70% confluent.

Replace the medium daily.

Replace the old growth mTeSR™1 Medium for 6 days.

(Optional) Prior to starting dissociation, add iROCK (10micromolar (µM)) in the 100-mm cell culture dish for 1h 0m 0s.

Coat the fresh 12-well plate with 6mL of cold Geltrex™ (0.5 µL/well) as described in steps 29-31.

Aspirate Geltrex™ and add 0.5 µL/well of antibiotic-free fresh mTeSR™1 Medium with iROCK (10micromolar (µM)).

Carefully aspirate the old growth medium from the cell 100-mm culture dishes.

Wash with 4 mL/dish of DPBS-/- and remove immediately.

Add 4mL/dish of Accutase™, ensure that cover of dish area.

Incubate the cell suspension at 37°C for 5-10 minutes.

Add 3mL/dish of DMEM/F12 and scrape the attached cells using a cell scraper.

Gently triturate the clusters across dish surface.

Transfer the unattached cell suspension using a 5 mL serological pipette into a 50 mL conical tube containing 10mL of DMEM/F12.

Spin down at 100x g,25°C.

Remove the supernatant and wash with 10mL of DPBS-/-.

Count the cells with Trypan blue dye exclusion.

Spin down at 100x g,25°C.

Aspirate and discard the supernatant.

Resuspend the cells with ice-cold DPBS-/-.

Gently mix the suspension cells and transfer 2 mL/15 mL conical tube (1x10⁶ cells per tube).

Spin down at 100x g,4°C.

Remove the supernatant and resuspend the pellet with 1SM electroporation buffer mix with the plasmids in a total volume of 100µL.

Immediately transfer the suspension cells into the 0.2 cm electroporation cuvette, preventing bubbles.

Electroporate the cells by using the Nucleofector 2B program A-23.

Immediately add 1mL of fresh mTeSR™1 Medium with iROCK (10micromolar (µM)) into the electroporation cuvette using a micropipette with a 1000 µL tip to avoid low cell viability.

Immediately transfer the cell suspension into a 15 mL conical tube containing 0.5 mL of antibiotic-free fresh mTeSR™1 Medium with iROCK (10micromolar (µM)).

Gently mix the cell suspension and transfer into three wells (0.5 mL/well) of the coated Geltrex™ 12-well plate previously prepared in steps 51-52.

Incubate the cells at 37°C, 95% O2, 5% CO₂ in humidified air.

After 24 h, replace the old growth medium with antibiotic-free fresh mTeSR™1 medium.

After 48 h of transfection, evaluate the integrase activity by flow cytometry.

Harvest cells using Accutase™ as described in step 24.

Remove the old growth medium and wash with 0.5mL/well of DPBS-/-, remove immediately.

Add 0.4mL/well of Accutase™.

Incubate the cells in the incubator at 37°C for 5-10 minutes.

Add 0.5mL/well of DMEM/F12 into a 15 mL conical tube containing 5mL of DMEM/F12.

Spin down at 100x g,25°C.

Aspirate and discard the supernatant.

Wash the cells with 2mL of ice-cold DPBS-/-.

Spin at 100x g,4°C.

Remove the supernatant and resuspend the cells in 2mL of ice-cold DPBS-/-.

Count the cells with Trypan blue dye exclusion.

Transfer 1 x 10⁵ cells/tube for flow cytometry analysis.

Keep the cell suspension in the original 15 mL conical tube and collect the pellet for DNA extraction.

Spin at 100x g,4°C.

Resuspend the cells (1 x 10⁵ cells/tube) in 300µL of ice-cold buffer for FACS.

Add 5µL of 7-AAD into a tube with the cells and incubate at Room temperature for 0h 10m 0s.

Culture NSC cells into a 60-mm culture dish until the cells cover the dish area, at 85-90% confluence. It is approximately 6-7 days.

Prior to starting the dissociation, coat the fresh 60-mm culture dishes with 3 mL/dish of cold Geltrex™ and place it into the incubator at 37°C for at least 0h 30m 0s.

Remove Geltrex™, and immediately add 2mL/dish of fresh NEM medium to prevent drying.

Place fresh culture dishes into the incubator at 37°C.

Carefully aspirate the old medium from the cell culture dish.

Wash the dish with 2mL of DPBS-/-, remove immediately.

Add 2mL of Accutase™ into the 60-mm culture dish, ensure that cover of dish area.

Incubate the cell suspension at 37°C for 3-5 minutes.

Add 3mL of DMEM/F12 and gently triturate the clusters across the dish surface.

Transfer the unattached cell suspension into a 15 mL conical tube containing 3mL of DMEM/F12 using a 5 mL serological pipette.

Spin down at 300x g,25°C.

Aspirate and discard the supernatant.

Wash with 5mL of DMEM/F12 using a 5 mL serological pipette.

Count the cells with Trypan blue dye exclusion.

Spin down at 300x g,25°C.

Resuspend the cell pellet with fresh growth NEM medium using a 5 mL serological pipette.

Add the cell suspension at 1 mL/dish into fresh 60-mm culture dishes previously prepared at steps 94-97. Should have a total volume of 3 mL/dish.

Incubate the cells at 37°C, 95% O2, 5% CO₂ in humidified air.

After 24 h, replace the old growth medium with 3mL of fresh NEM medium.

Split the cells every 6 days.

Carefully aspirate the old growth medium from the cell culture dish.

Replace with 4mL of fresh growth NEM medium.

Incubate the cells at 37°C, 95% O2, and 5% CO₂ in humidified air until they reach 80-90% confluent.

Change the growth medium every 2 days.

Replace the old growth NEM media for 6 days.

(Optional) Prior to starting dissociation, add iROCK (10micromolar (µM)) into 60-mm culture cells dish for 1h 0m 0s.

Coat the fresh 12-well plate with 6 mL of cold Geltrex™ (0.5 µL/well) as described in steps 95-97.

Aspirate Geltrex™ and add 0.5µL/well of antibiotic-free fresh NEM medium with iROCK (10micromolar (µM)).

Carefully aspirate the old growth medium from the cell 60-mm culture dishes.

Wash with 2mL/dish of DPBS-/-, and remove immediately.

Add 2mL/dish of Accutase™, ensure that cover of dish area.

Incubate the cell suspension at 37°C for 3-5 minutes.

Add 2mL/dish of DMEM/F12 and gently triturate the clusters across the dish surface.

Remove unattached cells using a 5 mL serological pipette.

Transfer the cell suspension into a 50 mL conical tube containing 10mL of DMEM/F12.

Spin down at 300x g,25°C.

Remove the supernatant and wash with 5mL of DPBS-/-.

Count the cells with Trypan blue dye exclusion.

Spin down at 300x g,25°C.

Resuspend the cells with ice-cold DPBS-/-.

Gently mix the suspension cells and transfer 2 mL/15 mL conical tube.

Spin down at 300x g,4°C.

Remove the supernatant and resuspend the cells with 1S electroporation buffer mix with the plasmids in a total volume of 100µL.

Immediately transfer the suspension cells into the 0.2 cm electroporation cuvette, avoid bubbles.

Electroporate the cells by using the Nucleofector 2D program A-33.

Immediately add 1mL of fresh NEM media with iROCK (10micromolar (µM)) into the electroporation cuvette using a micropipette with a 1000 µL tip to avoid low cell viability.

Immediately transfer the cell suspension into a 15 mL conical tube containing 0.5mL of antibiotic-free fresh NEM media with iROCK (10micromolar (µM)).

Gently mix the cell suspension and transfer into three wells (0.5 mL/well) of the coated Geltrex™ 12-well plate previously prepared in step 28.

Incubate the cells at 37°C, 95% O2, 5% CO₂ in humidified air.

After 24 h, replace the old growth medium with antibiotic-free fresh NEM medium.

After 48 h of transfection, the integrase activity can be evaluated by using flow cytometry.

Harvest cells using Accutase™.

Remove the old growth medium and wash with 0.5 mL/well of DPBS-/-, remove immediately.

Add 0.4mL/well of Accutase™.

Incubate the cells in the incubator at 37°C for 5-10 minutes.

Add 0.5mL/well of DMEM/F12 into a 15 mL conical tube containing 5mL of DMEM/F12.

Spin down at 300x g,25°C.

Wash the cells with ice-cold DPBS-/-.

Spin at 300x g,4°C.

Remove the supernatant and resuspend the cells in 2mL of ice-cold DPBS-/-.

Count the cells with Trypan blue dye exclusion.

Transfer 1 x 10⁵ cells/tube by flow cytometry analyses.

Keep the cell suspension in the original 15 mL conical tube and collect the pellet for DNA extraction.

Spin at 300x g,4°C.

Resuspend the cells (1 x 10⁵ cells/tube) in 300µL of ice-cold buffer for FACS.

Add 5µL of 7-AAD into a tube with the cells and incubate at Room temperature for 0h 10m 0s.