Alpha-1,3-galactosyltransferase-deficient miniature pigs produced byserial cloning using neonatal skin fibroblasts with loss ofheterozygosity

Collection with item attached
2017
Item details URL
http://open-repository.kisti.re.kr/cube/handle/open_repository/474437.do
DOI
10.5713/ajas.16.0010
Title
Alpha-1,3-galactosyltransferase-deficient miniature pigs produced byserial cloning using neonatal skin fibroblasts with loss ofheterozygosity
Description
This research was supported by a grant (PJ011375) from theNext-Generation BioGreen 21 Program, Rural Development Administration, agrant (2014034046) supported by the Bio & Medical Technology DevelopmentProgram, and a grant (2009-0093829) supported by the Priority ResearchCenters Program through National Research Foundation (NRF) funded by theMinistry of Science, ICT and Future Planning.
abstract
Objective: Production of alpha-1,3-galactosyltransferase (GT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous aGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce aGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts.
Methods: Miniature pig fibroblasts were transfected with aGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous aGT gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-alpha-1,3-galactose, an epitope produced by aGT. Using magnetic activated cell sorting, cells with monoallelic disruption of aGT were removed. Remaining cells with LOH carrying biallelic disruption of aGT were used for the second round NT to produce homozygous aGT gene-targeted piglets.
Results: Monoallelic mutation of aGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous aGT gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous aGT knockout piglets.
Conclusion: The present study demonstrates that the time required for the production of aGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial
provenance
Made available in Cube on 2018-09-28T10:46:02Z (GMT). No. of bitstreams: 0
language
English
author
Kim, Young June
Ahn, Kwang Sung
Kim, Minjeong
Kim, Min Ju
Ahn, Jin Seop
Ryu, Junghyun
Heo, Soon Young
Park, Sang-Min
Kang, Jee Hyun
Choi, You Jung
Shim, Hosup
accessioned
2018-09-28T10:46:02Z
available
2018-09-28T10:46:02Z
issued
2017
citation
ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES(30): 3
issn
1011-2367
uri
http://open-repository.kisti.re.kr/cube/handle/open_repository/474437.do
Funder
교육부
Funding Program
BK21플러스사업(0.5)
Project ID
1345273935
Jurisdiction
Rep.of Korea
Project Name
Global Research Center for Nanobiomedical Science in Regenerative Medicine
rights
openAccess
subject
Nuclear Transfer
Alpha-1,3-galactosyltransferase
Gene Targeting
Lossof heterozygosity
Pig
type
article


Files in This Item

There are no attached files.