TITLE: PiggyBac Transfection iPSC
AUTHORS: Aazam Vahdatshoar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Day before transfection: Re-plating the cells]
Aspirate the medium from one confluent well of 6-well plate, and wash once with PBS.
?. [Day before transfection: Re-plating the cells]
Add  accutase and leave in the incubator for .
1 mL
?. [Day before transfection: Re-plating the cells]
Add  growth medium or hES medium with a 1000p, and pipet up and down to dissociate the cells and break them into single cells.
1 mL
?. [Day before transfection: Re-plating the cells]
Transfer the cell suspension to a  conical tube. Centrifuge at  for . Remove the supernatant.
15 mL
Centrifuge: 800 33
?. [Day before transfection: Re-plating the cells]
Add  StemFlex to resuspend and count the cells with cell counter.
1 mL
?. [Day before transfection: Re-plating the cells]
Take out 6-well plate coated with Matrigel from  incubator after at least one-hour incubation.
37 °C
?. [Day before transfection: Re-plating the cells]
Aspirate the Matrigel carefully without scratching the surface. Wash once with PBS to remove residual Matrigel.
?. [Day before transfection: Re-plating the cells]
Plate 1.5 X 106 cells (when slow growers) in  StemFlex supplemented with  ROCK inhibitor in one well of Matrigelcoated 6-well plate.
2 mL
You may seed another well with fewer cells at 1.0 X 106 to compare the wells on the day of transfection to choose one. Cells should be about 70% confluent the next day.
?. [Day of transfection]
Prepare the transfection mix in  Eppendorf tubes under cell culture hood.
1.5 mL
?. [Day of transfection]
Add  Opti-MEM medium to the  tube for each transfection.
200 µl
1.5 mL
?. [Day of transfection]
Add  of the piggybac plasmid to the tube.
2 µg
?. [Day of transfection]
Add  of the transposase plasmid to the mix.
1.5 µg
?. [Day of transfection]
Add  TransIT-LT1 Transfection Reagent and mix well (ratio 1:3 DNA:Reagent).
10.5 µl
?. [Day of transfection]
Leave under the hood at  for .
0 Room temperature
?. [Day of transfection]
Remove the cell culture plate from day before and choose the well that is about 70-80% confluent in single layer cells.
?. [Day of transfection]
Remove the medium and feed with  StemFlex supplemented with  ROCK inhibitor (Y).
2 mL
?. [Day of transfection]
Add the  transfection mix evenly and drop-wise on the culture plate.
200 µl
?. [Day of transfection]
Shake the plate very gently to make sure it is dispersed evenly.
?. [Day of transfection]
Leave at  incubator for  without disturbing it.
37 °C
?. [Day of transfection]
Aspirate the medium and feed with  StemFlex + Y.
2 mL
?. [Day of transfection]
Change the medium for every day with StemFlex + Y.
?. [Day of transfection]
Two days after transfection add  blasticidin (BsD) if the backbone of the piggybac plasmid has the selection gene for blasticidin resistance. If cells look unhealthy after the transfection, do  blasticidin (BsD).
?. [Day of transfection]
Continue selection for  (max). Changing the medium every day with StemFlex +  Y +  BsD.
?. [Day of transfection]
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.
?. [Day of transfection]
You do not need to supplement StemFlex with ROCK inhibitor after day 7 of transfection, but do use RI at the following passage.
?. [Day of transfection]
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).
?. [Important QC tests on the newly generated polyclonal iPSC lines]
Expand a polyclonal line upfront at the lowest passage possible into a large batch.
?. [Important QC tests on the newly generated polyclonal iPSC lines]
Keep the passage as low as possible. Preferred to not use the line more than 5 passages after the Piggybac transfection.
If the line is stable and has good karyotype, if may be reconsidered to use the line at later passages.
?. [Important QC tests on the newly generated polyclonal iPSC lines]
Karyotype the newly generated line to determine any abnormal chromosomal events.
?. [Important QC tests on the newly generated polyclonal iPSC lines]
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.
?. [Important QC tests on the newly generated polyclonal iPSC lines]
For a monoclonal culture of your key cell line you may perform the standard 20 count cell karyotyping.
?. [Important QC tests on the newly generated polyclonal iPSC lines]
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.
It also shows the copy number for the number of transposon integrations (protocol available at the Khurana Lab/Aazam).NGN2-Puro-PB Construct Map: