Stable expression of a heterogeneous gene introduced via gene targeting into theHPRT locus of human fibrosarcoma cells

Abstract

To obtain a cell line that maintains stability of gene expression is important for industrial production of therapeutic proteins from recombinant cells. In this study, we attempted to improve the stability of expression of an exogenous gene by using the gene-targeting method in cultured cells. In our gene-targeting system, the green fluorescent protein (GFP) gene was used as an exogenous reporter gene targeted to the locus of the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene, which is constitutively expressed. Cell lines selected using markers of the targeting DNA were cultivated for 129 days without any drug selection, and the expression levels of GFP protein and the chromosomal structure of the gfp gene in these cell lines were evaluated. Cell lines in which gfp genes were randomly integrated into the genome showed decreased GFP expression, which resulted from loss of genes or attenuation of transcription. In contrast, cell lines in which the gfp gene was targeted to the hprt locus maintained a stable chromosomal structure and stable expression of the gfp gene, even after prolonged cultivation. These results suggest that constitutively expressed endogenous gene loci may be suitable positions for stable expression of exogenous genes, and that the gene-targeting strategy presented here may be useful for generation of cell lines for industrial protein production.

Generation of monoclonal antibody-producing mammalian cell lines

“…Cell line development using recombinase-mediated targeted integration has been shown to have advantages over random integration. More stable production and minimal clonal variations have been reported [154,155,159,160,168]. However, the mAb titer achieved is less than that achieved using random integration.…”

Dysregulation of the SIRT1/OCT6 Axis Contributes to Environmental Stress-Induced Neural Induction Defects

“…Considering the pattern of decreased Sirt1 expression during normal neural differentiation (Figures S4H and S4I), we revealed that the downregulation of SIRT1 deacetylase activity is a prerequisite for the efficient conversion of pluripotent stem cells to a neural fate during gastrulation. Through CRISPR/Cas9-mediated homologous recombination, we further integrated CAG promoter-driven wild-type Sirt1 or a deacetylase-inactive Sirt1 mutant ( Sirt1 H355A) into the Hprt locus for constitutive overexpression in mESCs (Cong et al., 2013, Koyama et al., 2006, Liu et al., 2016a). The overexpression efficiency was examined by qPCR and western blotting (Figures S4J and S4K).…”

Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins

“…Two different human gene loci, neither of which is regulated by HMGA1 proteins in the cell types being investigated (unpublished data), were analyzed for repair efficiency: the HPRT gene locus, which has previously been demonstrated to be constitutively expressed in normal somatic cells [10] and the β-globin gene locus, which is transcriptionally silent in all cells except those of erythroid lineage 11]. The cell lines used to assess repair efficiency in these two gene loci were the previously described transgenic HMGA1 "OFF" and HMGA1 "ON" cells [4], as well as non-transgenic MCF7 human breast epithelial cells and normal human embryonic fibroblasts (strain IMR90).…”

“…Utilizing a recently developed quantitative long-range PCR-based assay for measuring genespecific removal of UV-induced lesions [7], we assessed the efficiency of removal of UVinduced DNA damage in two different human genes: the hypoxanthine phosphoribosyl transferase (HPRT) gene, an allele of which is constitutively expressed in all normal somatic cells [8][9][10]), and the β-globin gene, which is transcriptionally silent in all cells but those of erythroid lineage [11]. These genes were chosen because microarray and other analyses indicated that HMGA1 proteins do not regulate the transcription of either gene in the cell lines being investigated (unpublished data), an important consideration given that these proteins can either positively or negatively regulate the transcription of a large number of genes in vivo (reviewed in: [12]).…”