| Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate. | |
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MedLine Citation:
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PMID: 20149191 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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BACKGROUND: The recent availability of alpha-1,3-galatosyltransferase knockout pigs has eliminated anti-Gal antibodies to the galalpha1-3gal (alphagal epitope) as the major barrier to xenotransplantation. These alphagal epitope-negative animals can also be produced by somatic cell nuclear transfer of cells overexpressing endo-beta-galactosidase (EndoGalC), an enzyme capable of digesting the alphagal epitope. For this, selection of cells with highly reduced synthesis of alphagal epitope is a prerequisite. In this study, we developed a novel method of selection using isolectin BS-I-B(4)-conjugated saporin (IB4-SAP), a targeted cytotoxin, that is specific for the terminal alphagal epitope. METHODS: A mixture of alphagal epitope-expressing and non-expressing pig cells was obtained by transfection with an EndoGalC expression vector. These cells were incubated with a solution containing IB4-SAP for 2 h at 37 degrees C, and subsequently cultivated for over 2 months under general conditions. RESULTS: Almost all (98%) of surviving cells were completely negative for expression of alphagal epitope, as confirmed by cytochemical staining using fluorescence-labeled IB4. FACS analysis also confirmed that the IB4-SAP-treated cells exhibited a staining pattern similar to that of the IB4-negative human cells. Extended cultivation (more than 6 months) of these IB4-SAP-treated cells did not alter the above staining pattern. RT-PCR analysis revealed the presence of EndoGalC mRNA in these cells. CONCLUSIONS: This IB4-SAP-mediated method of selection of alphagal epitope-negative cells will provide an alternative to the present method of cytotoxicity-based selection using specific antibody and complement. |
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Authors:
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Eri Akasaka; Satoshi Watanabe; Takehiro Himaki; Masato Ohtsuka; Mitsutoshi Yoshida; Kazuchika Miyoshi; Masahiro Sato |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Xenotransplantation Volume: 17 ISSN: 1399-3089 ISO Abbreviation: Xenotransplantation Publication Date: 2010 Jan-Feb |
Date Detail:
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Created Date: 2010-02-12 Completed Date: 2010-04-26 Revised Date: 2010-10-25 |
Medline Journal Info:
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Nlm Unique ID: 9438793 Medline TA: Xenotransplantation Country: Denmark |
Other Details:
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Languages: eng Pagination: 81-9 Citation Subset: IM |
Affiliation:
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Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Animals, Genetically Modified Cell Separation / methods* Cells, Cultured / drug effects* Cytotoxins / pharmacology* Epitopes / immunology Galactosyltransferases / genetics Humans Lectins / pharmacology* Ribosome Inactivating Proteins, Type 1 / pharmacology* Swine* Transplantation, Heterologous / immunology* |
| Chemical | |
Reg. No./Substance:
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0/Cytotoxins; 0/Epitopes; 0/IB4-saporin conjugate; 0/Lectins; 0/Ribosome Inactivating Proteins, Type 1; EC 2.4.1.-/Galactosyltransferases; EC 2.4.1.87/N-acetyllactosaminide alpha-1,3-galactosyltransferase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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