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

Asian-Australasian Journal of Animal Sciences

Deug-Nam Kwon¹  Jin-Hoi Kim¹  Ji Woo Kim²  Yeong Ji Kim²  Man-Jong Kang² 

[1] Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea;;

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

DOI  :  10.5713/ajas.15.0244

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown