Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity
Objective Production of alpha-1,3-galactosyltransferase (αGT)-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 αGT knockout pigs should be bred at least f...
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| Format: | Article |
| Language: | English |
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Asian-Australasian Association of Animal Production Societies
2017-03-01
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| Series: | Asian-Australasian Journal of Animal Sciences |
| Subjects: | |
| Online Access: | http://www.ajas.info/upload/pdf/ajas-30-3-439.pdf |
| _version_ | 1819032173764673536 |
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| author | Young June Kim Kwang Sung Ahn Minjeong Kim Min Ju Kim Jin Seop Ahn Junghyun Ryu Soon Young Heo Sang-Min Park Jee Hyun Kang You Jung Choi Hosup Shim |
| author_facet | Young June Kim Kwang Sung Ahn Minjeong Kim Min Ju Kim Jin Seop Ahn Junghyun Ryu Soon Young Heo Sang-Min Park Jee Hyun Kang You Jung Choi Hosup Shim |
| author_sort | Young June Kim |
| collection | DOAJ |
| description | Objective Production of alpha-1,3-galactosyltransferase (αGT)-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 αGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce αGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods Miniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous αGT 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-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were used for the second round NT to produce homozygous αGT gene-targeted piglets. Results Monoallelic mutation of αGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous αGT 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 αGT knockout piglets. Conclusion The present study demonstrates that the time required for the production of αGT-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 length of time for breeding. |
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| id | doaj.art-f62d7bee55684eb79232a9862ffdf5b0 |
| institution | Directory Open Access Journal |
| issn | 1011-2367 1976-5517 |
| language | English |
| last_indexed | 2024-12-21T06:57:44Z |
| publishDate | 2017-03-01 |
| publisher | Asian-Australasian Association of Animal Production Societies |
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| series | Asian-Australasian Journal of Animal Sciences |
| spelling | doaj.art-f62d7bee55684eb79232a9862ffdf5b02022-12-21T19:12:17ZengAsian-Australasian Association of Animal Production SocietiesAsian-Australasian Journal of Animal Sciences1011-23671976-55172017-03-0130343944510.5713/ajas.16.001023528Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosityYoung June Kim0Kwang Sung Ahn1Minjeong Kim2Min Ju Kim3Jin Seop Ahn4Junghyun Ryu5Soon Young Heo6Sang-Min Park7Jee Hyun Kang8You Jung Choi9Hosup Shim10 Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, KoreaObjective Production of alpha-1,3-galactosyltransferase (αGT)-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 αGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce αGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods Miniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous αGT 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-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were used for the second round NT to produce homozygous αGT gene-targeted piglets. Results Monoallelic mutation of αGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous αGT 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 αGT knockout piglets. Conclusion The present study demonstrates that the time required for the production of αGT-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 length of time for breeding.http://www.ajas.info/upload/pdf/ajas-30-3-439.pdfNuclear TransferAlpha-13-galactosyltransferaseGene TargetingLoss of heterozygosityPig |
| spellingShingle | Young June Kim Kwang Sung Ahn Minjeong Kim Min Ju Kim Jin Seop Ahn Junghyun Ryu Soon Young Heo Sang-Min Park Jee Hyun Kang You Jung Choi Hosup Shim Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity Asian-Australasian Journal of Animal Sciences Nuclear Transfer Alpha-1 3-galactosyltransferase Gene Targeting Loss of heterozygosity Pig |
| title | Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity |
| title_full | Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity |
| title_fullStr | Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity |
| title_full_unstemmed | Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity |
| title_short | Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity |
| title_sort | alpha 1 3 galactosyltransferase deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity |
| topic | Nuclear Transfer Alpha-1 3-galactosyltransferase Gene Targeting Loss of heterozygosity Pig |
| url | http://www.ajas.info/upload/pdf/ajas-30-3-439.pdf |
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