Figure 5

Development and validation of stably transfected polyclonal CHO-DG44 pools. (A) Schematic representation of IRES-DHFR cassette cloning using the overlapping PCR method. (B) Flow cytometry analysis of electroporated cells after 24 h of nucleofection. pMax-GFP vector was utilized as a transfection efficiency control. GFP positive cell count was detected as 96.9% (on the right). The flow cytometry result on the left indicates untransfected control with 0% GFP positive CHO-DG44 suspension cells. (C) Cell viability and (D) total cell count percentage during DHFR-based auxotrophic selection. In brief, cells were harvested in HT deprived CD OptiCHO selection media and Trypan blue exclusion-based counting was performed every two to three days. Cells were subcultured at 100.000–200.000 cells/ml. Taking into consideration the growth rate of CHO-DG44 pools, the cells were transferred from multiwell plates to orbital shake flasks. (E) The validation of genome integration was performed via conventional non-quantitative PCR using primer sets provided in Table 3. For the CAG promoter, the primer set was altered from P1–P3 pair to P2–P3 that amplifies a shorter fragment with lower GC content (see schematic). Specifically, while P3 reverse primer binds to a region at the 5’ end of the Fluc gene, P2 forward primer targets a region close to the 3’ end of the CAG promoter, that is further verified by Sanger sequencing (data not shown). Note that two different DNA ladders (1 kb DNA ladder—left and 1 kb plus DNA ladder—right) were used in the agarose gel experiments. Expected amplicon and relevant DNA ladder band sizes are highlighted on the agarose gel images. The schematics in (A) and (E) are created with BioRender.com.