MOUSE MODEL OF IGA DEFICIENCY USING GENE TARGETING

IGA缺乏症是人类最常见的体液免疫缺陷， 大约每600人中就有1人受到影响。这一缺陷导致了 血清或粘膜分泌物中几乎完全不存在可检测到的IgA 并与感染和自身免疫发病率的增加有关 疾病。目前尚不存在选择性IgA的小动物模型 缺乏症。这个项目的近期目标是开发一种鼠标 利用基因打靶功能缺失IgA缺陷模型的研究 小鼠免疫球蛋白重链(IgH)IgA基因座。越是长期的 这个项目的目标是深入了解IgA B细胞的个体发育和 免疫球蛋白A在系统和粘膜免疫反应中的作用这样的知识将 可能会增强我们对体液免疫缺陷的理解， 尤其是IgA缺乏症，应该会增强我们的设计能力 更好的口服和全身疫苗，以对抗各种病原体，如艾滋病毒。 此外，通过基因打靶获得了一只IgA缺陷小鼠 将进一步扩大利用基因打靶技术开发动物的经验 人类疾病的模型。该项目的近期目标将是 实现方式如下。来自小鼠品系的胚胎干细胞 129(古铜色)将具有IGHα恒定区外显子 使用同源重组技术删除。生产重组产品 含有这种缺失的小鼠，突变的胚胎干细胞将被 将其导入C57BL/6(黑毛色)囊胚，然后将其 植入养母体内。嵌合的雄性后代具有最大的 突变的胚胎干细胞的贡献，由 毛色嵌合程度，将与C57BL/6雌性小鼠配种。 突变的生殖系传递到F1世代将是 子代尾部DNA的Southern blotting鉴定。杂合子 将培育出纯合子小鼠以获得缺乏IgH IgA的纯合子小鼠 轨迹。确认IgH IgA基因座的突变导致了 通过测量血清中的IgA水平，将获得IgA表达的丢失 用IgA同型特异性酶联免疫吸附试验检测突变小鼠 脾B细胞表面IgA在Peyer‘s斑或脾B细胞上的表达 细胞学。将培育出免疫球蛋白A缺陷小鼠，建立一个 将可用于未来旨在评估B细胞的研究 个体发育和免疫球蛋白同型在黏膜组织中的表达 免疫球蛋白A在黏膜抗微生物免疫反应中的作用

IgA deficiency is the most common humoral immunodeficiency in humans, affecting approximately 1 in 600 people. This deficiency results in the virtual complete absence of detectable IgA in serum or mucosal secretions and is associated with an increased incidence of infections and autoimmune diseases. No small animal model currently exists for selective IgA deficiency. The immediate objective of this project is to develop a mouse model of IgA deficiency using gene targeting to functionally delete the immunoglobulin heavy chain (Igh) IgA locus from mice. The more long-term goal of this project is to gain insight into IgA B cell ontogeny and the role of IgA in systemic and mucosal immune responses. Such knowledge will likely enhance our understanding of humoral immunodeficiencies, particularly IgA deficiency, and should enhance our ability to design better oral and systemic vaccines against various pathogens, such as HIV. In addition, the production of an IgA deficient mouse by gene targeting will further expand the experience using gene targeting to develop animal models of human diseases. The immediate objective of this project will be achieved as follows. Embryonic stem cells derived from the murine strain 129 (agouti coat color) will have the Igh alpha constant region exons deleted using homologous recombination techniques. To produce recombinant mice containing this deletion, the mutated embryonic stem cells will be introduced into C57BL/6 (black coat color) blastocysts which will then be inserted into foster mothers. Chimeric male offspring with the largest contribution from the mutated embryonic stem cells, as determined by extent of coat color chimerism, will be bred with C57BL/6 female mice. Germline transmission of the mutation into the F1 generation will be assessed by Southern blotting of offspring tail DNA. Heterozygote offspring will be bred to obtain homozygous mice lacking the Igh IgA locus. Confirmation that mutation of the Igh IgA locus has resulted in loss of IgA expression will be obtained by measuring serum IgA levels in the mutant mice with an IgA isotype-specific ELISA and by measuring expression of surface IgA on Peyer's patch or spleen B cells with flow cytometry. The IgA deficient mice will be bred to establish a colony which will then be available for future studies intended to evaluate B cell ontogeny and immunoglobulin isotype expression in mucosal tissues and the role of IgA in mucosal immune responses against microbial pathogens.