title: p53 gene targeting by homologous recombination in fish ES cells.
abstract: BACKGROUND
  Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes).
  
  
  RESULTS
  Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo.
  
  
  CONCLUSIONS
  Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yan
authors.forename: Ni
authors.initials: N
authors.lastname: Hong
authors.forename: Tiansheng
authors.initials: T
authors.lastname: Chen
authors.forename: Mingyou
authors.initials: M
authors.lastname: Li
authors.forename: Tiansu
authors.initials: T
authors.lastname: Wang
authors.forename: Guijun
authors.initials: G
authors.lastname: Guan
authors.forename: Yongkang
authors.initials: Y
authors.lastname: Qiao
authors.forename: Songlin
authors.initials: S
authors.lastname: Chen
authors.forename: Manfred
authors.initials: M
authors.lastname: Schartl
authors.forename: Chang-Ming
authors.initials: CM
authors.lastname: Li
authors.forename: Yunhan
authors.initials: Y
authors.lastname: Hong
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D053595:Embryonic Stem Cells / Q000166:cytology*; D018390:Gene Targeting / Q000379:methods*; D016158:Genes, p53 / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D005838:Genotype; D059765:Homologous Recombination / Q000235:genetics*; D017403:In Situ Hybridization; D023421:Models, Animal; D009990:Oryzias / Q000235:genetics*