Prime Tech Ltd, Tsuchiura, Japan.
Xenotransplantation. 2009 Nov-Dec;16(6):511-21. doi: 10.1111/j.1399-3089.2009.00549.x.
For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of alphaGal expression by its digestion enzyme, endo-beta-galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross-breeding of pigs expressing EndoGalC and hDAF.
After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace x Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross-breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction.
Well-designed cloned pigs were produced by cross-breeding. alphaGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild-type pigs. hDAF expression reached about 10- to 70-fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti-pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week.
Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of alphaGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.
为了成功进行器官异种移植，人们积极地培育了基因工程猪。我们关注的重点是：（i）通过其消化酶内-β-半乳糖苷酶 C（EndoGalC）消化来减少αGal 的表达，以及（ii）通过人衰变加速因子（hDAF）抑制补体激活。细胞分选和核转移使高水平表达转基因的克隆猪的有效生产成为可能。我们报告了成功培育出表达 EndoGalC 和 hDAF 的猪的交叉繁殖。
在将 hDAF 和 EndoGalC 基因分别转染到来自长白猪 x 约克夏和梅山的胎儿的猪成纤维细胞中之后，使用细胞分选器收集有效表达转基因的转染细胞。使用体细胞核转移技术生产克隆猪。在克隆猪的杂交繁殖之后，将表达 EndoGalC 和 hDAF 的肾脏移植到狒狒中，以检验基因转导的效果。
通过杂交繁殖成功生产了经过精心设计的克隆猪。与野生型猪相比，克隆猪的αGal 表达水平降低了 2 到 14%。hDAF 的表达水平达到了人脐静脉内皮细胞的 10 到 70 倍。未观察到先天性畸形。没有发现死胎率增加或生长迟缓的问题。在未经任何抗猪抗体清除治疗的情况下，这种克隆猪到狒狒的肾脏移植中可以避免超急性排斥反应。然而，移植物在移植后 1 小时就出现纤维蛋白沉积，并在 1 周后被排斥。
使用细胞分选系统有效地收集转染细胞，我们生产了两种具有高水平 hDAF 表达和低水平αGal 表达的克隆猪。这种遗传修饰在预防超急性排斥反应方面是有效的，但在移植后立即出现促凝作用，导致急性血管排斥反应。即使高水平表达 hDAF，也有必要有效地抑制抗体与移植物的结合。
