title: Stable expression of a heterogeneous gene introduced via gene targeting into the HPRT 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.
authors.affiliation: Research Laboratory, TOTO Ltd., 2-1-1 Nakashima, Kokurakita-ku, Kita-kyushu 802-8601, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Koyama
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Banzai
authors.forename: Shuji
authors.initials: S
authors.lastname: Sonezaki
authors.forename: Kohji
authors.initials: K
authors.lastname: Kusano
mesh_terms: D001709:Biotechnology / Q000379:methods; D002460:Cell Line; D045744:Cell Line, Tumor; D005354:Fibrosarcoma / Q000378:metabolism*; D005786:Gene Expression Regulation; D018390:Gene Targeting; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase