Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

The overall goal of this procedure is to perform rapid and efficient targeted genome editing in the AAVS1 of human pluripotent stem cells, or human PSCs, using a flippase, Recombinase-mediated Cassette Exchange approach, or RMCE. RMCE improves the target insertion of a sequence into the AAVS1 locus, which is of great importance for stem cell biology as it allows rapid testing on the function of a molecule by overexpression or knockdown. The main advantage of this technique is that human PSC lines free of random integration events, still pluripotent and when normal karyotypes can be created in just 15 days.

This method provides a useful tool for transgenesis, in the AAVS1 locus but can also be applied to other human permissive loci. To begin, culture human PSC master cell lines under standard procedures on or off inactivated mouse embryonic fybroblasts or iMEFs, in six well plates. When the cells reach 60%to 70%confluency plate drug-resistant iMEFs by first using 0.1%gelatin solution to coat the necessary wells of a 12 well plate and incubate the plate at room temperature for five minutes.

Then, plate 125, 000 iDR4s per well and incubate the cultures overnight with iMEF medium. The following day, preincubate human PSC cultures with fresh medium including 10 micromolar ROCK Inhibitor at 37 degrees Celsius for one hour. In the meantime, bring human ESC transfection solution and the cell dissociation agent to room temperature.

Prepare one milliliter of nucleofection plating medium per nucleofection condition. Then, remove the iDR4 medium from the wells, use one milliliter of room temperature PBS to wash the cells and add nucleofection plating medium. Transfer 500 microliters per sample of plating medium to sterile 1.5 milliliter tubes and store at 37 degrees Celsius until plating.

To detach the human PSCs in a single cell suspension, remove the medium, use room temperature PBS to wash the cells and add one milliliter of 0.05%Trypsin or Accutase on feeder or feeder-free hPSC cultures, respectively. Incubate the cultures at 37 degrees Celsius. Next, carefully remove the plate from the incubator and without allowing the cells to detach, aspirate the dissociation agent and add 1 milliliter of human PSC medium.

Dissociate the loose cells by gentle flushing the medium on the plate surface while avoiding the formation of foam. Collect the cells in a single cell suspension in a clean and sterile 15 or 50 milliliter tube. Then use 1 milliliter of hESC medium to wash the cells and collect them in the same tube.

Take a 50 microliter aliquot of cell suspension for counting and centrifuge the remaining cells at 300 times g at room temperature for five minutes. Count the cells with a cell counting device while the suspension is undergoing centrifugation. Remove the supernatent and use room temperature PBS to gently resuspend the cell color to 10 to the sixth cell per milliliter.

Then transfer two milliliters per experimental condition to clean sterile 15 milliliter tubes and repeat the centrifugation. While the samples are spinning, prepare plasmid mixes in three clean 1.5 milliliter tubes with 0:1, 2:1 and 2:0 donor flippase molecular ratios using 2.5 micrograms of flippase plasmid. To proceed with nucleofection, remove the iDR4 plate and 1.5 milliliter tube with plating medium from the incubator.

One tube at a time, without disturbing the pellet, carefully pipette off the PBS, removing as much of the buffer as possible. Use 100 microliters of transfection solution to resuspend the pellet by gently pipetting. Then, transfer the cell suspension to the 1.5 milliliter tube containing the plasmid mixes and gently mix.

Transfer the cell DNA mix to the tranfector cuvette, paying attention not to introduce bubbles. Insert the cuvette in the nucleofector device and run program A13 for cells cultured on feeders or F16 for feeder-free transfection. Remove the cuvette and use the disposable transfer pipettes in the kit to add 0.5 milliliters of plating medium in the cuvette gently, but quickly and in one movement.

Then, recuperate the volume in the same manner and plate it drop-wise in the 12 well plate containing the iDR4 and plating medium. Place the culture dish on a shelf in the incubator and move it slowly back and forth and side to side to evenly distribute the cell suspension. Incubate the dish for 24 hours before changing the medium.

Two to three days posttransfection, start selection. Aspirate the medium, use 1 milliliter of PBS to wash the cells, then add human ESC medium with 100 nanograms per milliliter of puromycin. Observe cell growth and increase the puromycin concentration accordingly up to a maximum of 250 nanograms per milliliter, continuing puromycin selection for seven days.

After three to four days on puromycin selection, begin selection with 0.5 micromolar fialuridine or FIAU. Change the medium daily and maintain FIAU for no more than seven continuous days. After completion of selection, around days 14 to 15, resistant RMCE colonies will be present and constitute the new RMCE line.

Split the cells at a 1:2 ratio following standard procedures. When the wells from Key 1 are ready to split, dissociate the well for characterization into a single cell suspension as describe earlier in the video and collect the cells in a 15 milliliter tube. Spin down the cells at 300 times g at room temperature for five minutes.

Then, use one milliliter of PBS to resuspend the cells and split the sample in two. One filtered using a cell strainer to carry out flow cytometry and the other one for DNA analysis according to the text protocol. Following transfection with flippase recombinase and the tdT RMCE donor, hPSCs formed small groups of cells evenly distributed over the iDR4 MEF layer and expressed constitutive GFP and transient tdT.

Positive selection using puromycin favors RMCE donor insertion then negative selection using FIAU selects for only FRT mediated recombination events lacking TK.Around day six posttransfection, small clusters of RMCE GFP-negative, tdT-positive cells can be identified. As demonstrated here, GFP-negative, tdT-positive RMCE colonies are present only when both RMCE donor and flippase are used while RMCE does not occur in the absence of flippase. Flow cytometry characterization of the newly generated RMCE lines, which are the puromycin and FIAU resistant RMCE colonies, combined in a non-clonal cell population confirms that 100%of the cells represent the RMCE GFP-negative, tdT-positive phenotype.

When RMCE donors without recorder activity in hPSC are used, 100%of the cells are GFP-negative. PCR characterization shown here of the non-clonal RMCE line, demonstrates the full cassette exchange and that the selection program used generates random integration-free lines, both of the RMCE donor and the flippase expressing vector. In summary, this RMCE method significantly simplifies gene editing in hPSCs for a defined locus as compared to targeted nucleases.

Once mastered, this technique can be completed in 15 days. Therefore, RMCE is a very fast gene editing tool that will allow comparative studies in an isogenic setting. When using this procedure, it's important to have transfection efficiencies higher than 30%and to preform appropriate core storage selection agent.

After watching this video you should have a good understanding on how to perform RMCE in the AAVS1 locus of human PSCs and how to characterize the recombinate line.