English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

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 ).    Methodology and Principal Findings  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.

p53 Gene Targeting by Homologous Recombination in Fish ES Cells