Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate
Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate
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DOI：
10.1111/j.1399-3089.2010.00568.x
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发表时间：
2010-01-01
影响因子：
3.9
通讯作者：
Sato, Masahiro
中科院分区：
文献类型：
--
作者：
Akasaka, Eri;Watanabe, Satoshi;Sato, Masahiro
背景：最近获得的α-1，3-半乳糖基转移酶基因敲除猪已经消除了抗Gal抗体作为异种移植的主要障碍。这些α-半乳糖表位阴性的动物也可以通过体细胞核移植过表达内切-β-半乳糖苷酶(EndoGalC)的细胞产生，内切酶是一种能够消化α-半乳糖苷酶表位的酶。为此，选择α-半乳糖表位合成高度减少的细胞是先决条件。在这项研究中，我们建立了一种新的方法，利用异凝素BS-I-B-4偶联皂苷(IB4-SAP)，一种针对末端α-GAL表位的靶向细胞毒素。方法：用EndoGalC表达载体转染表达和不表达α-GAL表位的猪细胞。这些细胞与含有IB4-SAP的溶液在37℃孵育2小时，然后在普通条件下培养2个月以上。结果：几乎所有(98%)存活细胞的α-半乳糖表位表达完全阴性，荧光标记的IB4细胞化学染色证实了这一点。流式细胞仪分析还证实，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。长期培养(超过6个月)IB4-SAP处理的细胞不改变上述染色模式。结论：IB4-SAP介导的α-Gal表位阴性细胞的筛选方法将替代目前基于细胞毒作用的特异性抗体和补体筛选方法。
Background:The recent availability of alpha-1,3-galatosyltransferase knockout pigs has eliminated anti-Gal antibodies to the gal alpha 1-3gal (alpha gal epitope) as the major barrier to xenotransplantation. These alpha gal 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 alpha gal epitope. For this, selection of cells with highly reduced synthesis of alpha gal 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 alpha gal epitope.Methods:A mixture of alpha gal 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 alpha gal 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 alpha gal epitope-negative cells will provide an alternative to the present method of cytotoxicity-based selection using specific antibody and complement.