title: Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.
abstract: Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.
authors.affiliation: Department of Animal Science,College of Agriculture and Life Sciences,Chonnam National University,Gwangju,Korea.
authors.forename: Young-Hee
authors.initials: YH
authors.lastname: Jeong
authors.affiliation: Department of Animal Science,College of Agriculture and Life Sciences,Chonnam National University,Gwangju,Korea.
authors.forename: Yeong Ji
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Faculty of Biotechnology,College of Applied Life Sciences,Jeju National University,Jeju,Korea.
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Kim
authors.affiliation: Department of Animal Science,College of Agriculture and Life Sciences,Chonnam National University,Gwangju,Korea.
authors.forename: Se Eun
authors.initials: SE
authors.lastname: Kim
authors.affiliation: Department of Animal Science,College of Agriculture and Life Sciences,Chonnam National University,Gwangju,Korea.
authors.forename: Jiwoo
authors.initials: J
authors.lastname: Kim
authors.affiliation: Faculty of Biotechnology,College of Applied Life Sciences,Jeju National University,Jeju,Korea.
authors.forename: Min Jee
authors.initials: MJ
authors.lastname: Park
authors.affiliation: Department of Animal Biotechnology,KonKuk University,Seoul,Korea.
authors.forename: Hong-Gu
authors.initials: HG
authors.lastname: Lee
authors.affiliation: Department of Animal Science,College of Agriculture and Life Sciences,Chonnam National University,Gwangju,Korea,Faculty of Biotechnology,College of Applied Life Sciences,Jeju National University,Jeju,Korea.
authors.forename: Se Pill
authors.initials: SP
authors.lastname: Park
authors.affiliation: Department of Animal Science,College of Agriculture and Life Sciences,Chonnam National University,Gwangju,Korea,Faculty of Biotechnology,College of Applied Life Sciences,Jeju National University,Jeju,Korea.
authors.forename: Man-Jong
authors.initials: MJ
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D015153:Blotting, Western; D064113:CRISPR-Cas Systems; D002364:Caseins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D004720:Endonucleases / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D002364:Caseins; D016222:Fibroblast Growth Factor 2; D004720:Endonucleases
keywords: CRISPR/Cas9 system; Knock-in; Transgenic cattle; β-Casein gene