A TRANSGENIC PIG MODEL FOR SICKLE CELL DISEASE

本项目的目的是建立一个转基因猪模型， 镰状细胞病没有自然发生的非人类同源， 疾病和动物模型的可用性将大大提高 我们研究疾病和开发新疗法的能力。猪 为人体循环生理学提供了一个通常很好的模型， 与人类相似的某些方面， 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，以及毛细血管中的其他相似性 床密度、某些红细胞结构特性和 血管疾病以前尝试制造转基因小鼠 镰状细胞病的模型还没有产生令人满意的 动物学，可能是由于鼠和人之间的差异 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， 在猪身上产生疾病的必要条件， 用于生产正常人血红蛋白的高效构建， 转基因猪，已经组装合成血红蛋白， 从LCR和人α和β基因产生，并诱变 包括成人β-珠蛋白基因产生镰刀突变和两个 额外的突变，安的列斯和D旁遮普，这促进镰刀 血红蛋白聚合此外，为了便于维护和 我们生产的转基因猪的临床分析，我们正在开发一种 一种非常小的猪种，Panepinto微型猪，作为一种转基因猪， 系统采用标准的超数排卵方案， 大小的猪，我们已经获得了显着的超数排卵（高达约50 排卵数/头猪）和受精卵回收（高达约20个/头猪） 自然繁殖后。对照DNA结构已经被 显微注射，以开发DNA导入程序，外科手术 再植入和维持妊娠，这将是必要的 将转基因技术扩展到这种更方便的大小品种。我们 了解和治疗镰状细胞病的能力应该大大提高， 由于精确的转基因猪模型的可用性， 易于进行临床评价。

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.