Document Detail


Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate.
MedLine Citation:
PMID:  20149191     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Eri Akasaka; Satoshi Watanabe; Takehiro Himaki; Masato Ohtsuka; Mitsutoshi Yoshida; Kazuchika Miyoshi; Masahiro Sato
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Xenotransplantation     Volume:  17     ISSN:  1399-3089     ISO Abbreviation:  Xenotransplantation     Publication Date:    2010 Jan-Feb
Date Detail:
Created Date:  2010-02-12     Completed Date:  2010-04-26     Revised Date:  2010-10-25    
Medline Journal Info:
Nlm Unique ID:  9438793     Medline TA:  Xenotransplantation     Country:  Denmark    
Other Details:
Languages:  eng     Pagination:  81-9     Citation Subset:  IM    
Affiliation:
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
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MeSH Terms
Descriptor/Qualifier:
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:
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

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