title: Construction of a novel expression system on a human artificial chromosome.
abstract: Efficient regulation of transgene would greatly facilitate the analysis of gene function in biological systems for basic research and clinical applications. The tetracycline-regulatable system (TRS) has proven to be a promising tool for such purposes. Despite their widespread application, a number of challenges are still associated with the use of TRS, including clonal variability in the regulation and copy number. We have recently constructed a novel human artificial chromosome (HAC) called 21DeltaqHAC. By housing a TRS-based DNA-PKcs expression cassette in this HAC, we were able to circumvent the problems associated with conventional TRS-based vectors. We achieved tight control of DNA-PKcs expression and rescued the radiosensitive phenotype of DNA-PKcs-deficient CHO cells. The combined use of HAC and the TRS serves as a model for controllable and fixed copy number expression vectors. Our study also demonstrates the suitability of the HAC to accommodate multi-subunit constructs such as that of the TRS.
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medical Sciences, Tottori University, 86 Nishicho, Yonago, Tottori 683-8503, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Otsuki
authors.forename: Candice G T
authors.initials: CG
authors.lastname: Tahimic
authors.forename: Nozomi
authors.initials: N
authors.lastname: Tomimatsu
authors.forename: Motonobu
authors.initials: M
authors.lastname: Katoh
authors.forename: David J
authors.initials: DJ
authors.lastname: Chen
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kurimasa
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
mesh_terms: D000818:Animals; D016466:CHO Cells; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases