Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
Xenotransplantation. 2010 Jan-Feb;17(1):81-9. doi: 10.1111/j.1399-3089.2010.00568.x.
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.
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.
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.
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.
最近产生的α-1,3-半乳糖基转移酶基因敲除猪消除了抗 Gal 抗体对 galalpha1-3gal（Gal 抗原表位）的作用，从而成为异种移植的主要障碍。这些缺乏 Gal 抗原表位的动物也可以通过表达内-β-半乳糖苷酶（EndoGalC）的体细胞细胞核移植产生，该酶能够消化 Gal 抗原表位。为此，选择 Gal 抗原表位合成高度降低的细胞是先决条件。在这项研究中，我们开发了一种使用伴刀豆球蛋白 A（ConA）结合蓖麻毒素（IB4-SAP）的新型选择方法，该方法是一种针对末端 Gal 抗原表位的靶向细胞毒素。
通过用 EndoGalC 表达载体转染获得表达和不表达 Gal 抗原表位的猪细胞混合物。将这些细胞在 37°C 下用含有 IB4-SAP 的溶液孵育 2 小时，然后在一般条件下培养超过 2 个月。
用荧光标记的 IB4 进行细胞化学染色证实，几乎所有（98%）存活的细胞完全缺乏 Gal 抗原表位的表达。流式细胞术分析也证实，IB4-SAP 处理的细胞表现出与 IB4 阴性的人类细胞相似的染色模式。对这些经过 IB4-SAP 处理的细胞进行长时间（超过 6 个月）培养不会改变上述染色模式。RT-PCR 分析显示这些细胞中存在 EndoGalC mRNA。
这种基于 IB4-SAP 的 Gal 抗原表位阴性细胞选择方法将为目前使用特异性抗体和补体的细胞毒性选择方法提供替代方法。
