PiggyBac Transfection iPSC
Aazam Vahdatshoar
Abstract
This protocol details the generation of stably transfected iPSC with piggybac constructs. This procedure is used to introduce exogenous NGN2 gene into iPSCs. The established cell lines are used for trans-differentiation into neurons by doxycycline induction.
Attachments
Steps
Day before transfection: Re-plating the cells
Aspirate the medium from one confluent well of 6-well plate, and wash once with PBS.
Add 1mL accutase and leave in the incubator for 0h 2m 0s.
Add 1mL growth medium or hES medium with a 1000p, and pipet up and down to dissociate the cells and break them into single cells.
Transfer the cell suspension to a 15mL conical tube. Centrifuge at 800rpm,0h 0m 0s for 0h 3m 0s. Remove the supernatant.
Add 1mL StemFlex to resuspend and count the cells with cell counter.
Take out 6-well plate coated with Matrigel from 37°C incubator after at least one-hour incubation.
Aspirate the Matrigel carefully without scratching the surface. Wash once with PBS to remove residual Matrigel.
Plate 1.5 X 106 cells (when slow growers) in 2mL StemFlex supplemented with 10micromolar (µM) ROCK inhibitor in one well of Matrigelcoated 6-well plate.
Day of transfection
Prepare the transfection mix in 1.5mL Eppendorf tubes under cell culture hood.
Add 200µL Opti-MEM medium to the 1.5mL tube for each transfection.
Add 2µg of the piggybac plasmid to the tube.
Add 1.5µg of the transposase plasmid to the mix.
Add 10.5µL TransIT-LT1 Transfection Reagent and mix well (ratio 1:3 DNA:Reagent).
Leave under the hood at Room temperature for 0h 20m 0s.
Remove the cell culture plate from day before and choose the well that is about 70-80% confluent in single layer cells.
Remove the medium and feed with 2mL StemFlex supplemented with 10micromolar (µM) ROCK inhibitor (Y).
Add the 200µL transfection mix evenly and drop-wise on the culture plate.
Shake the plate very gently to make sure it is dispersed evenly.
Leave at 37°C incubator for 6h 0m 0s without disturbing it.
Aspirate the medium and feed with 2mL StemFlex + Y.
Change the medium for every day with StemFlex + Y.
Two days after transfection add 5µg/ml blasticidin (BsD) if the backbone of the piggybac plasmid has the selection gene for blasticidin resistance. If cells look unhealthy after the transfection, do 2.5µg/ml blasticidin (BsD).
Continue selection for 120h 0m 0s (max). Changing the medium every day with StemFlex + 10micromolar (µM) Y + 5µg/ml BsD.
If you see more than 80% of cells die before 5 days, stop selection and feed the cells with StemFlex without BsD until it becomes confluent for expansion.
You do not need to supplement StemFlex with ROCK inhibitor after day 7 of transfection, but do use RI at the following passage.
Expand the cell line (use RI during the passaging) and freeze a large number of tubes. When passaging the cells for the 1st time, add RI to the media.
Important QC tests on the newly generated polyclonal iPSC lines
Depending on the experiment, you may choose to keep the line as polyclonal or monoclonal by subcloning it and select for single clones to use moving forward (2-3 clones for QC and you can pool or keep separate).
Expand a polyclonal line upfront at the lowest passage possible into a large batch.
Keep the passage as low as possible. Preferred to not use the line more than 5 passages after the Piggybac transfection.
Karyotype the newly generated line to determine any abnormal chromosomal events.
For most purposes karyotyping the polyclonal line on 50 cells to get a sense of the karyotypically unstable population (if any) is fine. Karyotype the lowest passage available.
For a monoclonal culture of your key cell line you may perform the standard 20 count cell karyotyping.
Perform the Splinkerette PCR assay to locate and map the exact location of each transposition event in the genome of the cells that are stably transfected with piggybac construct.
