A TRANSGENIC PIG MODEL FOR SICKLE CELL DISEASE

该项目的目的是生产一种转基因猪模型，用于 镰状细胞病。没有自然产生的非人类同源词 这种疾病和动物模型的可用性将极大地提高 我们研究这种疾病和开发新疗法的能力。那头猪 为人类循环生理学提供了一个总体上很好的模型， 具有与人类相似的某些方面，尤其与 该模型例如对血管活性化合物的类似反应，类似的 对细胞外血红蛋白的反应，以及毛细血管的其他相似之处 床层密度，某些红细胞结构性质和发生 脑血管疾病。之前制造转基因小鼠的尝试 镰状细胞病的模型尚未产生令人满意的结果 症状学，可能是由于小鼠和人类之间的差异 血管解剖学和生理学。 为了达到人类镰状血红蛋白(HBS)的高水平 在我们利用最多的猪身上产生疾病所必需的 用于生产正常人血红蛋白的高效结构 已组装成合成血红蛋白的转基因小猪 从LCR和人类的α和β基因产生，以及诱变 包括产生镰状突变的成人β-珠蛋白基因和两个 其他突变，安的列斯和D旁遮普，促进镰刀 血红蛋白聚合。此外，为了方便维护和 我们生产的转基因猪的临床分析，我们正在开发一种 非常小的猪品系，Panepinto微型猪，作为转基因猪 系统。使用为FULL的超数排卵开发的标准方案 大小的猪，我们已经获得了显著的超数排卵(高达50头左右 每头猪排卵)和受精卵回收(每头猪最多20个) 在自然繁殖之后。对照DNA的构建已经被 显微注射以开发引入DNA的程序，外科 再次植入和维持妊娠，这是必要的 将转基因技术推广到这种体型更方便的品种。我们的 了解和治疗镰状细胞病的能力应该很强 准确的转基因猪模型的出现加速了这一进程， 易于接受临床评估。

The purpose of this project is to produce a transgenic pig model for sickle cell disease. There is no naturally occurring non-human cognate to the disease and the availability of an animal model would greatly enhance our ability to study the disease and develop new therapies. The pig provides a generally excellent model for human circulatory physiology, with certain aspects of similarity to humans of particular relevance to the model such as analogous responses to vaso-active compounds, a similar response to extracellular hemoglobin, and other similarities in capillary bed density, certain red cell structural properties and the occurrence of cerebrovascular diseases. Previous attempts to make a transgenic mouse model for sickle cell disease have not produced satisfactory symptomatology, probably due to differences between murine and human vascular anatomy and physiology. In order to achieve the high levels of human sickle hemoglobin (HbS) necessary to produce the disease in pigs we are utilizing the most efficient construction for the production of normal human hemoglobin in transgenic lull size pigs that has been assembled for synthetic hemoglobin production from the LCR and human alpha and beta genes, and mutagenizing the included adult beta-globin gene to produce the sickle mutation and two additional mutations, Antilles and D Punjab, which promote sickle hemoglobin polymerization. In addition, to facilitate the maintenance and clinical analysis of the transgenic pigs we produce, we are developing a very small strain of pig, the Panepinto miniature swine, as a transgenic system. Using standard protocols developed for the superovulation of full size pigs, we have obtained significant superovulation (up to about 50 ovulations/pig) and recovery of fertilized ova (up to about 20/pig) following natural breeding. Control DNA constructions have been microinjected to develop procedures for the introduction of DNA, surgical reimplantation and maintenance of pregnancy which will be necessary to extend transgenic technology to this more conveniently sized breed. Our ability to understand and treat sickle cell disease should be greatly accelerated by the availability of an accurate transgenic pig model, readily amenable to clinical evaluation.