Genomic DNA extraction and Junction PCR
Genomic DNAs were purified from pellets of KI cell pools using GeneJET
Genomic DNA Purification Kit (Thermo Fisher Scientific) and served as
templates for PCR using the Phusion High-Fidelity PCR Master Mix (Thermo
Fisher Scientific). To verify TI, 5’ Junction PCR was conducted by
touchdown PCR (98°C for 30 s; 10×: 98°C for 10 s, 72–62°C
[−1°C/cycle] for 30 s, 72°C for 45 s [short variant of CMV,
EF-1α], 1 min [CHEF-1α]; 30×: 98°C for 10 s, 62°C for 30 s, 72°C
for 45 s [short variant of CMV, EF-1α], 1 min [CHEF-1α]; 72°C
for 10 min). For the generation of double KI monitoring CHO-K1 cell
line, out-out and 5’/3’ Junction PCR was performed by touchdown PCR
(98°C for 30 s; 10×: 98°C for 10 s, 70–60°C [−1°C/cycle] for 30 s,
72°C for 2 min 10 s [out-out], 1 min 20 s [5’ Junction], 1 min
[3’ Junction]; 30×: 98°C for 10 s, 60°C for 30 s, 72°C for 2 min 10
s [out-out], 1 min 20 s [5’ Junction], 1 min [3’ Junction];
72°C for 10 min). PCR primers are listed in Supplementary Table S3.