Cell culture and transfection
The adherent CHO-K1 cell lines were maintained in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) FBS (Hyclone).
Cells were maintained as monolayer cultures in 25 cm2
T-flasks (Nunc) with a working volume of 5 ml. The serum free suspension
adapted CHO-K1 cells were maintained in Power CHO medium (Lonza)
supplemented with 8 mM L-Glutamine (Hyclone) and cultivated as
suspension culture in 125 ml Erlenmeyer flasks (Corning) with a working
volume of 20 ml. All cells were incubated in a humidified 5%
CO2 at 37°C without shaking (adherent), or with 120 rpm
shaking (suspension). Viable cell concentration was measured by a
Countess II FL automated cell counter (Invitrogen) with the trypan
blue dye exclusion method. Two transfection methods were used in this
study; electroporation and lipofection. Electroporation was conducted
using NEPA21 electroporator (Nepagene, Japan). According to the
manufacturer’s instructions, adherent CHO-K1 cells were washed twice
with Opti-MEM (Gibco) but not serum-free suspension CHO-K1.
1.0×106 cells were pelleted and re-suspended in 100 µl
mixture of Opti-MEM (Gibco) and 10 µg DNA of Cas9/sgRNA expression
vector and donor plasmid at a ratio 1:1 (w:w). Poring pulse and transfer
pulse condition were as follows: 135 V, 7.5 miliseconds (ms) pulse
length, two pulses, 50 ms pulse interval, 10% decay rate with +
polarity and 20 V, 50 ms pulse length, five pulses, 50 ms pulse
interval, 40% decay rate with +/- polarity respectively. After the
electroporation, cells were immediately seeded in 6 well plates with
pre-warmed culture media. Lipofection was conducted using Lipofectamine
3000 (Invitrogen) as per the manufacturer’s instructions.