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Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs
- Source :
- PLoS ONE, PLoS ONE, Vol 8, Iss 12, p e84250 (2013)
- Publication Year :
- 2013
-
Abstract
- Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.
- Subjects :
- Swine
Science
Xenotransplantation
medicine.medical_treatment
Biology
Animals, Genetically Modified
Gene Knockout Techniques
Plasmid
medicine
Animals
Gene
Alleles
Transcription activator-like effector nuclease
Multidisciplinary
Base Sequence
Gene targeting
Fibroblasts
Galactosyltransferases
Molecular biology
Phenotype
Cell culture
Gene Targeting
Mutation
Medicine
Somatic cell nuclear transfer
Swine, Miniature
Research Article
Subjects
Details
- ISSN :
- 19326203
- Volume :
- 8
- Issue :
- 12
- Database :
- OpenAIRE
- Journal :
- PloS one
- Accession number :
- edsair.doi.dedup.....426f7bb5ab4755d82c93780dc2d2637b