30. U6 promoter forward primer (Table 2).
31. Temperature-controlled water bath.
32. Presto™ Mini Plasmid Kit, Geneaid™ Midi Plasmid Kit
(Geneaid).
2.3 Gene Targeting
of Eβ-iPSCs
1. Amaxa 4D-nucleofector System (Lonza).
2. P3 Primary Cell 4D-Nucleofector™ X Kit S (Lonza). This kit
contains Nucleofector™ Solution, Supplement, and 16-well
Nucleocuvette™ Strips (20 μL).
3. sgRNA/PX459 plasmid, used for gene targeting of the Eβ-iPSCs: construction described in Subheading 3.3.
4. Single-stranded DNA oligonucleotide (ssODN) repair template. The ssODN repair template for HbE correction contains
the homology arms of 90 nucleotides on either side of the
point mutation (a total of 181 nucleotides).
(a) Order the ssODN repair template according to the
sequence below (see Note 1). The nucleotide “G” (bold
letter) represents the right nucleotide. CAAACAGACAC
CATGGTGCATCTGACTCCTG
AGGAGAAGTCTGCCGTTACTGCCC
TGTGGGGCAAGGTGAACGTGGAT
GAAGTTGGTGGTGAGGCCCTGGGCAGGTTGG
TATCAAGGTTACAAGACA
GGTTTAAGGAGACCAATAGAAACTG
GGCATGTGGAGACAGAGAAGACTCTTGGG.
(b) Resuspend the ssODN to 200 μM in sterile ddH 2 O and
store at À20
C.
2.4 Clonal Isolation
of Genetic Corrected
iPSCs
1. 40-μm cell strainer.
2. 96-well plates.
3. SMC4 small molecule cocktail (Corning).
2.5 Verification
of Clones by Multiplex
PCR Analysis
1. Heat block.
2. HotStarTaq polymerase, PCR buffer, MgCl 2 , dNTPs
(Qiagen).
3. Primers for multiplex PCR analysis for HbE mutation
(Table 3).
Table 2
Primer sequence for U6 promoter
Primer name
Oligo sequence (5
0
! 3
0 )
Length (base)
Ref.
U6-forward primer
GAGGGCCTATTTCCCATGATTCC
23
[11]
198
Methichit Wattanapanitch
31. Temperature-controlled water bath.
32. Presto™ Mini Plasmid Kit, Geneaid™ Midi Plasmid Kit
(Geneaid).
2.3 Gene Targeting
of Eβ-iPSCs
1. Amaxa 4D-nucleofector System (Lonza).
2. P3 Primary Cell 4D-Nucleofector™ X Kit S (Lonza). This kit
contains Nucleofector™ Solution, Supplement, and 16-well
Nucleocuvette™ Strips (20 μL).
3. sgRNA/PX459 plasmid, used for gene targeting of the Eβ-iPSCs: construction described in Subheading 3.3.
4. Single-stranded DNA oligonucleotide (ssODN) repair template. The ssODN repair template for HbE correction contains
the homology arms of 90 nucleotides on either side of the
point mutation (a total of 181 nucleotides).
(a) Order the ssODN repair template according to the
sequence below (see Note 1). The nucleotide “G” (bold
letter) represents the right nucleotide. CAAACAGACAC
CATGGTGCATCTGACTCCTG
AGGAGAAGTCTGCCGTTACTGCCC
TGTGGGGCAAGGTGAACGTGGAT
GAAGTTGGTGGTGAGGCCCTGGGCAGGTTGG
TATCAAGGTTACAAGACA
GGTTTAAGGAGACCAATAGAAACTG
GGCATGTGGAGACAGAGAAGACTCTTGGG.
(b) Resuspend the ssODN to 200 μM in sterile ddH 2 O and
store at À20
C.
2.4 Clonal Isolation
of Genetic Corrected
iPSCs
1. 40-μm cell strainer.
2. 96-well plates.
3. SMC4 small molecule cocktail (Corning).
2.5 Verification
of Clones by Multiplex
PCR Analysis
1. Heat block.
2. HotStarTaq polymerase, PCR buffer, MgCl 2 , dNTPs
(Qiagen).
3. Primers for multiplex PCR analysis for HbE mutation
(Table 3).
Table 2
Primer sequence for U6 promoter
Primer name
Oligo sequence (5
0
! 3
0 )
Length (base)
Ref.
U6-forward primer
GAGGGCCTATTTCCCATGATTCC
23
[11]
198
Methichit Wattanapanitch