title: Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein.
abstract: Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Sang Mi
authors.initials: SM
authors.lastname: Lee
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Ji Woo
authors.initials: JW
authors.lastname: Kim
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Young-Hee
authors.initials: YH
authors.lastname: Jeong
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Se Eun
authors.initials: SE
authors.lastname: Kim
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Yeong Ji
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Seung Ju
authors.initials: SJ
authors.lastname: Moon
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Ji-Hye
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Keun-Jung
authors.initials: KJ
authors.lastname: Kim
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Kim
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea .
authors.forename: Man-Jong
authors.initials: MJ
authors.lastname: Kang
keywords: Homologous Recombination; Knock-in; Porcine β-casein Gene; Therapeutic Proteins