A multiauxotrophic CHO cell line for the rapid isolation of producers of diverse or high levels of recombinant proteins

Chinese Hamster Ovary (CHO) cells are widely used for the high-level production of recombinant proteins. We created a multiauxotrophic mutant of CHO-K1 cells, CHO8A, that is deficient in eight enzymatic steps in the purine/pyrimidine biosynthetic pathways. Prototrophy was restored by transfections with complementary DNA-based genes for the eight missing activities. CHO8A cells permit: (1) selection of transfectant clones that have incorporated genes for eight or more different polypeptides, suitable for engineering complex proteins, or pathways; and (2) the single-step selection of high producers of a particular protein. The latter is achieved by simultaneous use of eight vectors, each bearing one of the eight rescue genes and a cargo protein gene. Screening as few as 10 surviving colonies yielded high producers secreting mAbs at 84 picograms per cell per day or more. CHO8A was isolated by CRISPR-Cas9 knockout of 10 genes in the pathways to pyrimidines (Dhodh, Umps, Ctps1, Ctps2, and Tyms) and purines (Paics, Atic, Impdh1, Impdh2, and Gmps).

Automated summary

CHO8A, a multiauxotrophic mutant of CHO-K1 cells, was created by restoring the prototrophy of the cells through transfection of complementary DNA-based genes. This cell line allows for the selection of transfectant clones with multiple polypeptide genes, as well as the single-step selection of high producers of a specific protein.