Applied StemCell, Inc., Milpitas, California, United States of America.
PLoS One. 2019 Jul 25;14(7):e0219842. doi: 10.1371/journal.pone.0219842. eCollection 2019.
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.
哺乳动物细胞表达系统是生产生物治疗蛋白最常用的平台。然而，由于随机整合导致的转基因表达不稳定和可变性，重组哺乳动物细胞系的开发常常受到阻碍。我们开发了一种高效的基因定点整合策略。该方法能够在哺乳动物细胞的特定位点快速、精确地插入基因表达盒，从而实现均一的转基因表达。我们确定 Hipp11（H11）基因为 CHO-S 细胞中基因敲入的“安全港”位点。利用成簇规律间隔短回文重复序列（CRISPR）/Cas9 介导的同源重组，我们敲入了一个包含一对 PhiC31 噬菌体 attP 位点和促进基于整合酶的 GOI 整合的基因的 DNA 盒（着陆垫）。建立了一个带有正确插入 H11 基因座的着陆垫的主细胞系。该主细胞系用于 PhiC31 整合酶催化的特异性、不可逆重组。使用该系统，在选择后 GFP 的整合效率达到 97.7%。然后，使用类似的方案，在 HEK293T 细胞中在 ROSA26 基因座插入 GFP 基因，进一步验证了该系统，选择后 GFP 阳性细胞的比例为 90.7%。相比之下，随机插入在 CHO-S 和 HEK293T 细胞中分别产生了 0.68%和 1.32%的 GFP 阳性细胞。总之，这些发现证明了一种准确有效的产生重组细胞系的方案，以提供一致的蛋白质生产。在 CHO-S 和 HEK293T 两种细胞系中，使用两个不同的基因组位点作为整合位点，证明了其广泛的适用性。因此，该系统对于生物制造治疗蛋白可能具有重要价值。
