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Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination
- Source :
- Asian-Australasian Journal of Animal Sciences, Vol 29, Iss 4, Pp 564-570 (2016), Asian-Australasian Journal of Animal Sciences
- Publication Year :
- 2016
- Publisher :
- Asian-Australasian Association of Animal Production Societies, 2016.
-
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.
- Subjects :
- 0301 basic medicine
Transcription Activator-like Effector Nuclease [TALEN]
Nuclease
Transcription activator-like effector nuclease
Knockout rat
biology
Effector
Electroporation
Knockout
Sal-like 1 Gene
lcsh:Animal biochemistry
Molecular biology
Article
03 medical and health sciences
030104 developmental biology
biology.protein
Animal Science and Zoology
lcsh:Animal culture
Homologous recombination
Homologous Recombination
Gene
lcsh:QP501-801
Gene knockout
Food Science
lcsh:SF1-1100
Subjects
Details
- Language :
- English
- ISSN :
- 19765517 and 10112367
- Volume :
- 29
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- Asian-Australasian Journal of Animal Sciences
- Accession number :
- edsair.doi.dedup.....558564e27090d38e4eb326949f38d03a