Disruption of the gene C12orf35 leads to increased productivities in recombinant CHO cell lines

Abstract

Recently, we reported that the loss of a telomeric region of chromosome 8 in Chinese Hamster Ovary (CHO) cells correlates with higher recombinant productivities. New cell lines lacking this region, called CHO-C8DEL, showed several advantages during cell line generation and for the production of recombinant proteins (Ritter et al., 2016, Biotechnol Bioeng). Here, we performed knock-down and knock-out experiments of genes located within this telomeric region of chromosome 8 to identify the genes causing the observed phenotypes of CHO-C8DEL cell lines. We present evidence that loss or reduced expression of the gene C12orf35 is responsible for higher productivities and shorter recovery times during selection pressure. These effects are mediated by increased levels of mRNA of the exogenes heavy chain (HC) and light chain (LC) as well as dihydrofolate reductase (DHFR) and neomycin phosphotransferase (Neo) during the stable expression of antibodies. Biotechnol. Bioeng. 2016;113: 2433-2442. © 2016 Wiley Periodicals, Inc.

Fam60A plays a role for production stabilities of recombinant CHO cell lines

“…Assuming that due to the high amino acid sequence conservation of 97% between human and hamster FAM60A protein the function and interaction with HDACs is conserved, the knock-out of Fam60A or the deletion of Fam60A as in the CHO-C8DEL cell lines might increase production stabilities of cell lines through a lack of HDAC inhibition, leading to increased histone acetylation and therefore to open and active chromatin states. However, we do not observe an increase of mRNA levels of HC and LC after repression of the gene Fam60A (Ritter et al, 2016b). However, we do not observe an increase of mRNA levels of HC and LC after repression of the gene Fam60A (Ritter et al, 2016b).…”

“…In comparison to our previous study in a mAb producing clone, which identified that the loss of the gene C12orf35 is causing higher productivities (Ritter et al, 2016b). Conversely, no increases in mRNA levels of HC and LC were detected after the repression of gene Fam60A in the analyzed mAb producing clones and pools (Ritter et al, 2016b). Flow cytometry analysis of mAb expressing pools (project A or project B) reveal changes in the fluorescence distribution not only after transfection of siRNAs against the gene C12orf35, but also after repression of the gene Fam60A ( Fig.…”

“…Each designed TALEN gene was synthetized, cloned, and transiently transfected in CHO-K1a cells as previously described (Ritter et al, 2016b). Each TALEN binds to 25 nucleotides on either the 5 0 (on the sense) or the 3 0 (on the antisense) DNA strand, respectively.…”

“…We showed that screening for the absence of genes located in this region is a powerful tool to identify high and stable producing clones in a fast and efficient way (Ritter et al, 2016a). We demonstrated that the loss of the gene C12orf35 is causing shorter recovery times during selective pressure using methotrexate (MTX) as well as higher productivities of CHO-C8DEL cell lines by increased mRNA levels of exogenes (Ritter et al, 2016b). Moreover, several fold higher volumetric pool productivities after transient and stable transfections were achieved and significantly more high and stable producing clones were subsequently recovered (Ritter et al, 2016a).…”

“…S1) and subsequently stably transfected with a vector encoding for a mAb (project A) as previously described (Ritter et al, 2016b). Two CHO-K1a clones with frame shift mutations resulting in an early stop codon within the first 120 bp of the gene Fam60A (CHO-K1a is haploid for the telomeric region of chromosome 8) were isolated ( Fig.…”