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DOI QR Code

Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination

  • Kim, Se Eun ;
  • Kim, Ji Woo ;
  • Kim, Yeong Ji ;
  • Kwon, Deug-Nam ;
  • Kim, Jin-Hoi ;
  • Kang, Man-Jong
  • Received : 2015.03.20
  • Accepted : 2015.08.07
  • Published : 2016.04.01

Abstract

The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using $300{\mu}g/mL$ G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene.

Keywords

Knockout;Homologous Recombination;Transcription Activator-like Effector Nuclease [TALEN];Sal-like 1 Gene

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Acknowledgement

Supported by : Rural Development Administration