Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs
Pigs are ideal organ donors for xenotransplantation and an excellent model for studying human diseases, such as neurodegenerative disease. Transcription activator-like effector nucleases (TALENs) are used widely for gene targeting in various model animals. Here, we developed a strategy using TALENs...
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Published in | Scientific reports Vol. 4; no. 1; p. 6926 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
London
Nature Publishing Group UK
05.11.2014
Nature Publishing Group |
Subjects | |
Online Access | Get full text |
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Summary: | Pigs are ideal organ donors for xenotransplantation and an excellent model for studying human diseases, such as neurodegenerative disease. Transcription activator-like effector nucleases (TALENs) are used widely for gene targeting in various model animals. Here, we developed a strategy using TALENs to target the
GGTA1
,
Parkin
and
DJ-1
genes in the porcine genome using Large White porcine fibroblast cells without any foreign gene integration. In total, 5% (2/40), 2.5% (2/80) and 22% (11/50) of the obtained colonies of fibroblast cells were mutated for
GGTA1
,
Parkin
and
DJ-1
, respectively. Among these mutant colonies, over 1/3 were bi-allelic knockouts (KO) and no off-target cleavage was detected. We also successfully used single-strand oligodeoxynucleotides to introduce a short sequence into the
DJ-1
locus. Mixed
DJ-1
mutant colonies were used as donor cells for somatic cell nuclear transfer (SCNT) and three female piglets were obtained (two were bi-allelically mutated and one was mono-allelically mutated). Western blot analysis showed that the expression of the DJ-1 protein was disrupted in KO piglets. These results imply that a combination of TALENs technology with SCNT can efficiently generate bi-allelic KO pigs without the integration of exogenous DNA. These
DJ-1
KO pigs will provide valuable information for studying Parkinson's disease. |
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ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/srep06926 |