title: Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination.
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 μg/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.
authors.affiliation: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors.forename: Se Eun
authors.initials: SE
authors.lastname: Kim
authors.affiliation: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors.forename: Ji Woo
authors.initials: JW
authors.lastname: Kim
authors.affiliation: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors.forename: Yeong Ji
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors.forename: Deug-Nam
authors.initials: DN
authors.lastname: Kwon
authors.affiliation: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors.forename: Jin-Hoi
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors.forename: Man-Jong
authors.initials: MJ
authors.lastname: Kang
keywords: Homologous Recombination; Knockout; Sal-like 1 Gene; Transcription Activator-like Effector Nuclease [TALEN]