Generation of KI monitoring cell lines
The promoter-trap based KI monitoring CHO-K1 cell line harboring
promoter-less EGFP expression cassette was generated in the previous
study (Lee et al., 2018). Briefly, targeted integrants expressing GFP in
the non-coding region of the CHO-K1 genome were transfected with two
sgRNA expression vectors targeting entire CMV promoter regions, upstream
of EGFP coding sequences, and then targeted clonal cells were isolated
(Lee et al., 2018). The double KI monitoring CHO-K1 cell line was
generated from the aforementioned single KI monitoring cell line. The
single KI monitoring cell line was co-transfected with Cas9/sgRNA
expression vector targeting COSMC locus and donor plasmid
containing puromycin and promoter-less TagRFP657 expression cassette as
described in Figure S4A. After two weeks of puromycin selection, a
limiting dilution was conducted for single cell cloning. The selected
clones were screened by out-out and 5’/3’ Junction PCR (Figure S4B), and
maintained in culture media with puromycin (3 µg/ml) and G418 (500
µg/ml). The adherent cell lines were adapted to serum free suspension
culture. Cells were seeded at 5.0×105 cells/ml in
6-well clear flat bottom not treated cell culture plate (Falcon) with
suspension culture media, Power CHO medium (Lonza) supplemented with 8
mM L-Glutamine (Hyclone). The fetal bovine serum (FBS, Hyclone)
gradually decreased from 3% to 0% until viable cell density reached up
to 1.0 – 1.5 ×106 cells/ml.