Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination
| Autor: | Se Eun Kim, Ji Woo Kim, Yeong Ji Kim, Deug-Nam Kwon, Jin-Hoi Kim, Man-Jong Kang |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: | |
| Zdroj: | Asian-Australasian Journal of Animal Sciences, Vol 29, Iss 4, Pp 564-570 (2016) |
| Druh dokumentu: | article |
| ISSN: | 1011-2367 1976-5517 |
| DOI: | 10.5713/ajas.15.0244 |
| Popis: | 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. |
| Databáze: | Directory of Open Access Journals |
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