3. Perform plasmid extraction using 50 mL of culture with Geneaid™ Midi Plasmid Kit according to the manufacturer’s
instruction (see Note 8).
4. Measure the plasmid concentration using NanoDrop 2000 and
store the plasmid at À20
C. This preparation will be used for
the nucleofection step in Subheading 3.4.
5. Store the culture in 50% glycerol at À80
C.
3.4 Gene Targeting
of Eβ-iPSCs
Genetic correction of HbE mutation in the Eβ-iPSCs is performed
using the sgRNA/PX459 plasmid generated in Subheading 3.3.3
and the ssODN repair template (Subheading 2.3) on the Amaxa
4D-Nucleofector System. The protocol is adapted from previously
described steps [11, 12].
1. Culture the Eβ-iPSCs in mTeSR™1 medium on a Matrigelcoated 6-well plate (see Note 3) for 3–4 days or until they reach
50–70% confluence.
2. On the day of transfection, pre-treat the Eβ-iPSCs with 10 μM
Y-27632 for an hour.
3. Prepare a Matrigel-coated 24-well plate for seeding (see Note
3).
4. Remove the mTeSR™1 medium from the 6-well cultured plate
(step 1) and wash the cells once with DPBS.
5. Add 1 mL/well Accutase and incubate at 37
C for 7 min.
6. Add 1 mL/well mTeSR™1 medium supplemented with 10
μM Y-27632, count the cells (200,000 cells per reaction is
required), centrifuge the cells in a 15-mL conical tube at 500
 g for 5 min at room temperature.
7. Prepare the P3 Primary Cell Nucleofector™ solution (Subheading 2.3, step 2) by mixing 3.6 μL of the supplement
with 16.4 μL of the Nucleofector™ solution in a 1.5-mL
microfuge tube.
8. Remove the medium (step 6) completely and resuspend
200,000 cells in 20 μL of the P3 Primary Cell Nucleofection™
solution (step 7).
9. Add 2 μg of the sgRNA/pX459 plasmid and 200 pmol ssODN
repair template (total volume does not exceed 2 μL) into the
cell suspension.
10. Transfer the mixture to one well of the 16-well Nucleocuvette™ Strip (see Note 9) and run the program CB-150 according to the manufacturer’s instruction.
11. Immediately add 80 μL of mTeSR™1 medium supplemented
with 10 μM Y-27632 into the well and transfer the cells into
the 24-well Matrigel-coated plate (from step 3) containing 1
mL of pre-warmed mTeSR™1 medium supplemented with 10
μM Y-27632.
204
Methichit Wattanapanitch
instruction (see Note 8).
4. Measure the plasmid concentration using NanoDrop 2000 and
store the plasmid at À20
C. This preparation will be used for
the nucleofection step in Subheading 3.4.
5. Store the culture in 50% glycerol at À80
C.
3.4 Gene Targeting
of Eβ-iPSCs
Genetic correction of HbE mutation in the Eβ-iPSCs is performed
using the sgRNA/PX459 plasmid generated in Subheading 3.3.3
and the ssODN repair template (Subheading 2.3) on the Amaxa
4D-Nucleofector System. The protocol is adapted from previously
described steps [11, 12].
1. Culture the Eβ-iPSCs in mTeSR™1 medium on a Matrigelcoated 6-well plate (see Note 3) for 3–4 days or until they reach
50–70% confluence.
2. On the day of transfection, pre-treat the Eβ-iPSCs with 10 μM
Y-27632 for an hour.
3. Prepare a Matrigel-coated 24-well plate for seeding (see Note
3).
4. Remove the mTeSR™1 medium from the 6-well cultured plate
(step 1) and wash the cells once with DPBS.
5. Add 1 mL/well Accutase and incubate at 37
C for 7 min.
6. Add 1 mL/well mTeSR™1 medium supplemented with 10
μM Y-27632, count the cells (200,000 cells per reaction is
required), centrifuge the cells in a 15-mL conical tube at 500
 g for 5 min at room temperature.
7. Prepare the P3 Primary Cell Nucleofector™ solution (Subheading 2.3, step 2) by mixing 3.6 μL of the supplement
with 16.4 μL of the Nucleofector™ solution in a 1.5-mL
microfuge tube.
8. Remove the medium (step 6) completely and resuspend
200,000 cells in 20 μL of the P3 Primary Cell Nucleofection™
solution (step 7).
9. Add 2 μg of the sgRNA/pX459 plasmid and 200 pmol ssODN
repair template (total volume does not exceed 2 μL) into the
cell suspension.
10. Transfer the mixture to one well of the 16-well Nucleocuvette™ Strip (see Note 9) and run the program CB-150 according to the manufacturer’s instruction.
11. Immediately add 80 μL of mTeSR™1 medium supplemented
with 10 μM Y-27632 into the well and transfer the cells into
the 24-well Matrigel-coated plate (from step 3) containing 1
mL of pre-warmed mTeSR™1 medium supplemented with 10
μM Y-27632.
204
Methichit Wattanapanitch