title: A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene.
abstract: Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Konishi
authors.forename: Sivasundaram
authors.initials: S
authors.lastname: Karnan
authors.forename: Miyuki
authors.initials: M
authors.lastname: Takahashi
authors.forename: Akinobu
authors.initials: A
authors.lastname: Ota
authors.forename: Lkhagvasuren
authors.initials: L
authors.lastname: Damdindorj
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hosokawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Konishi
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002934:Cinnamates / Q000494:pharmacology; D003001:Cloning, Molecular; D000229:Dependovirus / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005434:Flow Cytometry; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D000900:Anti-Bacterial Agents; D002934:Cinnamates; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase