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Updated: Jun 16, 2026
Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy
Published on: February 26, 2021
Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate
Eri Akasaka1, Satoshi Watanabe, Takehiro Himaki
1Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
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.
