Abstract
Mammalian cell expression systems are the most commonly used platforms for producing biotherapeutic proteins. However, development of recombinant mammalian cell lines is often hindered by the unstable and variable transgene expression associated with random integration. We have developed an efficient strategy for site-specific integration of genes of interest (GOIs). This method enables rapid and precise insertion of a gene expression cassette at defined loci in mammalian cells, resulting in homogeneous transgene expression. We identified the Hipp11 (H11) gene as a "safe harbor" locus for gene knock-in in CHO-S cells. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated homologous recombination, we knocked in a DNA cassette (the landing pad) that includes a pair of PhiC31 bacteriophage attP sites and genes facilitating integrase-based GOI integration. A master cell line, with the landing pad inserted correctly in the H11 locus, was established. This master cell line was used for site-specific, irreversible recombination, catalyzed by PhiC31 integrase. Using this system, an integration efficiency of 97.7% was achieved with green fluorescent protein (GFP) after selection. The system was then further validated in HEK293T cells, using an analogous protocol to insert the GFP gene at the ROSA26 locus, resulting in 90.7% GFP-positive cells after selection. In comparison, random insertion yielded 0.68% and 1.32% GFP-positive cells in the CHO-S and HEK293T cells, respectively. Taken together, these findings demonstrated an accurate and effective protocol for generating recombinant cell lines to provide consistent protein production. Its likely broad applicability was illustrated here in two cell lines, CHO-S and HEK293T, using two different genomic loci as integration sites. Thus, the system is potentially valuable for biomanufacturing therapeutic proteins.
Highlights
Over several decades, the Chinese hamster ovary (CHO) cell has served as the major standard expression platform for producing recombinant therapeutic proteins [1, 2]
The robust and ubiquitous function of H11 has been demonstrated in mice, pigs, human embryonic stem cells, and induced pluripotent stem cells [17,18,19]
Based on data from transgenic mice, human stem cells and pigs [17,18,19], we hypothesized that this region would serve as a "safe harbor" to provide robust and active transgene expression in CHO-S cells
Summary
The Chinese hamster ovary (CHO) cell has served as the major standard expression platform for producing recombinant therapeutic proteins [1, 2]. The traditional approach for developing recombinant CHO cell lines is random insertion of the gene of interest into the genome, followed by a selection for cells carrying the transgene [1, 2]. Site-specific integration of transgenes in mammalian cells
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