title: Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells.
abstract: BACKGROUND
  Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs) in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs) but poorly in hESCs.
  
  
  RESULTS
  Human ribosomal DNA (rDNA) repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA) gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50%) and homologous recombination frequency (>10(-5)) were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs.
  
  
  CONCLUSIONS
  This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs.
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Xionghao
authors.initials: X
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Li
authors.forename: Junlin
authors.initials: J
authors.lastname: Yang
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Xue
authors.forename: Youjin
authors.initials: Y
authors.lastname: Hu
authors.forename: Mai
authors.initials: M
authors.lastname: Feng
authors.forename: Wenbin
authors.initials: W
authors.lastname: Niu
authors.forename: Qiurui
authors.initials: Q
authors.lastname: Yang
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
authors.forename: Jiahui
authors.initials: J
authors.lastname: Xia
authors.forename: Lingqian
authors.initials: L
authors.lastname: Wu
authors.forename: Desheng
authors.initials: D
authors.lastname: Liang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004275:DNA, Ribosomal / Q000235:genetics* / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology*; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D056426:Genetic Loci; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D014162:Transfection / Q000379:methods
chemical_list: D004275:DNA, Ribosomal; D005164:Factor IX