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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

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random integration methods hesc regenerative medicine rdna locus of hescs mouse embryonic stem ribosomal dna rdna repeats problems method gene targeting transgenes in hescs physiological gene human coagulation factor ix 45s prerna rrna gene homologous recombination frequency human genetic research

Junlin Yang,Zhuo Li,Ming Lei,Yong Wu,Lingqian Wu,Xionghao Liu,Jinfeng Xue,Youjin Hu,Mai Feng,Wenbin Niu,Qiurui Yang,Jiahui Xia,Desheng Liang,.Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells.. 7 (5),.

Junlin Yang,Zhuo Li,Ming Lei,Yong Wu,Lingqian Wu,Xionghao Liu,Jinfeng Xue,Youjin Hu,Mai Feng,Wenbin Niu,Qiurui Yang,Jiahui Xia,Desheng Liang,"Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells." 7

Junlin Yang,Zhuo Li,Ming Lei,Yong Wu,Lingqian Wu,Xionghao Liu,Jinfeng Xue,Youjin Hu,Mai Feng,Wenbin Niu,Qiurui Yang,Jiahui Xia,Desheng Liang,"Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells."