Remodeling of glycoantigen in xenotransplantation

异种移植中糖抗原的重塑

• 批准号: 11557086
• 负责人: MIYAGAWA Shuji
• 金额: $ 8.58万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557086/
• 关键词: α1-3GT; Knockout; Targeting vector; Cre-loxP system; Codon; Transgenic mouse; Transgenic pig; GnT-III

The knockout (KO) of the α1,3 galactosyl transferase (α1,3GT) has been of considerable interest. However, the KO of this gene might well have other harmful effects on swine. Two lines of mice earring theα1,3GT targeting vector with three IoxP sites were produced. Two kinds of the transgenic mice with Cre recombinase gene, pCX-Cre and pLIV-Cre, were also obtained. The established conditional homologous recombination system was then tested. However, the efficiency of the recombination, via the Cre-/loxP system leaved considerable room for improvement.On the other hand, we synthesized "mammalian Cre recombinase gene" and examined its expression in Chinese hamster ovarian tumor (CHO) cells. This gene recombined the loxP sites much more efficiently than the wild-type Cre recombinase gene.Next, the effect of the various glycosyltransferases on glycosphingolipids was examined, using transfecte swine endothelial cell (SEC) lines. The overexpression of all two different types of glycosyltransferase, N-acetylglucosarninyl transferase III (GnT-III), as well as α2,6-sialyltransferase, α2,3-sialyltransferase andα1,2-fucosyltransferase, suppress the total antigenicity of SEC significantly. The levels of theα-galactosyl opitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, andα1,2FT transfectants.Finally, we have been successful in generating several lines of transgenic mice and pigs that contain the human GnT-III gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the α-galactosyl epitope

α1,3半乳糖转移酶（α 1,3gt）的敲除（KO）引起了相当大的兴趣。然而，这种基因的KO很可能对猪有其他有害影响。制备了具有3个IoxP位点的α 1,3gt靶向载体的2株小鼠。同时获得了两种Cre重组酶基因转基因小鼠pCX-Cre和pLIV-Cre。对所建立的条件同源重组体系进行了测试。然而，通过Cre-/loxP系统重组的效率仍有很大的改进空间。另一方面，我们合成了“哺乳动物Cre重组酶基因”，并检测了其在中国仓鼠卵巢肿瘤（CHO）细胞中的表达。该基因比野生型Cre重组酶基因更有效地重组了loxP位点。接下来，利用转染猪内皮细胞（SEC）系，研究了各种糖基转移酶对鞘糖脂的影响。过表达两种不同类型的糖基转移酶n -乙酰葡萄糖氨基转移酶III （GnT-III）以及α2,6-唾液基转移酶、α2,3-唾液基转移酶和α1,2- focusyltransferase均可显著抑制SEC的总抗原性。在鞘糖脂的水平α半乳糖opitope并不减少GnT-III转染子但ST6Gal我,ST3Gal三世和α1、2英尺转染子。最后，我们已经成功地培育了几种含有人类GnT-III基因的转基因小鼠和猪。小鼠和猪的GnT-III基因过表达降低了它们对人类天然抗体的抗原性，尤其是α-半乳糖表位

项目成果

Koyota S, Ikeda Y, Miyagawa S, et al.: "Downregulation of the alfa-Gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene: Modulation of the biosynthesis of terminal structures by a

Koyota S、Ikeda Y、Miyakawa S 等人：“通过转染 N-乙酰氨基葡萄糖转移酶 III 基因，下调猪内皮细胞 N-聚糖中的 alfa-Gal 表位表达：通过 a 基因调节末端结构的生物合成。”

^*S.Miyagawa, H.Murakami, A.Murase et al.: "Transgenic pigs with human N-acetylglucosaminyltransferase III"Transplant Proc.. 33. 742-743 (2001)

^*S.Miyakawa、H.Murakami、A.Murase 等人：“带有人 N-乙酰葡糖胺基转移酶 III 的转基因猪”Transplant Proc.. 33. 742-743 (2001)

Yukie Koresawa, Shuji Miyagawa, Masahito Ikawa, et al.: "A synthesis for new Cre recombinase gene based on optimal codon usage for mammalian systems"J. Biol. 127. 367-372 (2000)

Yukie Koresawa、Shuji Miyakawa、Masahito Ikawa 等人：“基于哺乳动物系统最佳密码子使用的新 Cre 重组酶基因的合成”J.

M. Koma, S. Miyagawa, S. Koyota, et al.: "Effect of Gal 1,4GlcNAc6- -D Sialyl Transferase on the swine xenoantigen"Transplant Proc. 32. 2509-2510 (2000)

M. Koma、S. Miyakawa、S. Koyota 等人：“Gal 1,4GlcNAc6- -D 唾液酸转移酶对猪异种抗原的影响”移植程序。

S Miyagawa, H Murakam, Y Takahagi et al.: "Remodeling of the Major Pig Xenoantigen by N-acetylglucosaminyltransferase III in Transgenic Pig"J Biol Chem. 276(42). 9310-9319 (2001)

S Miyakawa、H Murakam、Y Takahagi 等人：“转基因猪中 N-乙酰氨基葡萄糖转移酶 III 对主要猪异种抗原的重塑”J Biol Chem。