Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate.
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.
Akasaka
Eri
E
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
Watanabe
Satoshi
S
Himaki
Takehiro
T
Ohtsuka
Masato
M
Yoshida
Mitsutoshi
M
Miyoshi
Kazuchika
K
Sato
Masahiro
M
D000818:Animals; D030801:Animals, Genetically Modified; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured / Q000187:drug effects*; D003603:Cytotoxins / Q000494:pharmacology*; D000939:Epitopes / Q000276:immunology; D005700:Galactosyltransferases / Q000235:genetics; D006801:Humans; D037102:Lectins / Q000494:pharmacology*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000494:pharmacology*; D000076985:Saporins; D013552:Swine; D014183:Transplantation, Heterologous / Q000276:immunology*
D003603:Cytotoxins; D000939:Epitopes; C496198:IB4-saporin conjugate; D037102:Lectins; D054789:Ribosome Inactivating Proteins, Type 1; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D000076985:Saporins