3.2 Nucleofection of iPSCs

In order to electroporate piggyBac and transposase vectors into iPSCs in suspension, use the X-Unit of the 4D- Nucleofector™ System in combination with the P3 Primary Cell 4D-Nucleofector™ X Kit according to the manufacturer’s guidelines.

First of all, prepare the DNA, the Nucleofector™ solution and the cell culture plates. For a nucleofection reaction in 100 μl cuvettes, mix 10µg and 2.5µg in less than 10µL volume (maximum 10% of the final sample volume) in a 1.5 ml tube.

In a separate tube, mix 82µL with 18µL per nucleofection reaction and bring to Room temperature. Prepare Matrigel-coated cell culture plates with the desired volume of mTeSR™1 medium with ROCKi and prewarm in the incubator ( see Note 11 ).

Switch on the X-Unit of the 4D-Nucleofector™ System and choose the cell-type specific program for the human embryonic stem cell line H9, the cuvette size, P3 primary solution and the pulse CB-156 or CB-150 ( see Note 12 ).

Dissociate the cells to be nucleofected using TrypLE, centrifuge (400x g) and resuspend in mTeSR™1 with ROCKi.

Determine the cell number, transfer 800,000 cells for each nucleofection into a 1.5 ml tube and centrifuge (400x g).

Aspirate the supernatant and resuspend the cells in 100µL, mix with the DNA and transfer into an electroporation cuvette and close the lid. Avoid air bubbles while pipetting. Gently tap the cuvette to make sure that the sample covers the bottom.

Quickly put the cuvette(s) into the Nucleofector™ and press the start button to apply the pulse CB-156 or CB-150.

Immediately after, carefully remove the samples, add mTeSR™1 with ROCKi into the cuvette, mix by gently pipetting up and down two to three times and transfer the complete solution onto a Matrigel-coated plate with prewarmed medium and place in the incubator ( see Note 13 ).

The next day, wash the cells with 1x and change the medium to mTeSR™1 w/o ROCKi. Change the medium every day until next passaging ( see Fig. 2b). Starting 48h 0m 0s after nucleofection, select the cells with an integrated construct with the appropriate antibiotic ( see Note 14 ).

In order to determine the number of the integrated piggyBac constructs, use the piggyBac copy number kit from System Biosciences ( see Note 15 ). To prepare genomic DNA, seed the cells in a 12-well plate ( see Note 16 ). When confluent, wash once with 1x and add 250µL to each well.

Freeze the cells at -80°C and thaw the plate at Room temperature to ensure complete cellular lysis.

Detach the cells by pipetting up and down, transfer the lysates to 1.5 ml tubes and heat them at 95°C for 0h 2m 0s.

Centrifuge at 17000x g and transfer the supernatant to a new 1.5 ml tube. The lysates should be placed On ice if used immediately or stored at -20°C.

Prepare two master mixes of 4.75µL, 6.25µL, and 0.5µL per sample (one master mix with UCR1 primers for genomic DNA detection and one with PBcopy primers for piggyBac detection).

Aliquot 12µL per well of a 96-well plate and add 0.5µL (≤500ng). Seal the plate, carefully, mix by vortexing and briefly spin down.

Run the qPCR with the following program:

0h 2m 0s at 50°C,

0h 10m 0s at 95°C,

40 cycles of 95°C for 0h 0m 15s and 60°C for 0h 1m 0s,

followed by 0h 0m 15s at 95°C,

0h 0m 15s at 60°C, and 0h 0m 15s at 95°C ( see Note 17 ).

Calculate the copy number as follows [11]:

ΔΔC_t = 2^{(average PBcopy Ct–average UCR1 Ct)}, divide the ΔΔCt by 2 as there are two copies of the UCR1 sequence per genome.