Human Germline Ig Loci in Cloned Transgenic Cattle: Diversification & Immunophysiology

克隆转基因牛中的人类种系 Ig 位点：多样化

• 批准号: 0131335
• 负责人: Richard Goldsby
• 金额: $ 33万
• 依托单位: Amherst College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-15 至 2006-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0131335&HistoricalAwards=false
• 关键词: Human; Germline; Ig; Loci; Cloned

It has been possible to construct a human artificial chromosome (HAC) containing the entire unrearranged human heavy and light chain Ig loci and introduce it into a bovine fibroblast. Using advances in the technology of cloning, it has been possible to use these HAC-transgenic fibroblasts to generate bovine fetuses and calves. These cloned transgenic bovines will provide the experimental material for a project that will determine the ability of HAC-borne human Ig loci to undergo rearrangement, Ig expression and the generation of a primary antibody repertoire within the environment of bovine cells. While we hypothesize that a diverse repertoire of human antibodies will be formed in these animals, whether the bovine B lineage environment will support the generation of a diverse repertoire of human antibodies is an open question. There are major differences in the mechanisms and sites used by humans and Bovidae to diversify antibody genes. There is no body of experiment that predicts whether the rearrangement of HAC-borne human Ig loci will be limited, as is true of bovine Ig loci, or extensive, as seen in humans and transgenic mice. This experiment will allow us to investigate the interplay of species-specific cell physiology with the sequences of entire unrearranged immunoglobulin loci in determining the mechanism, extent and sites of immunoglobulin gene diversification. The availability of bovines transgenic for the complete human heavy chain locus and the complete human light chain locus make it possible to examine the compatibility of unrearranged Ig loci from one species with the diversification system of another. Although this question has been explored in genetic chimeras (i.e. 'human to mouse') between species that use the same mechanisms and sites of repertoire diversification, it has not been studied in those that do not. The creation of 'human to bovine' Ig locus chimeras provides an opportunity to determine the extent to which human Ig loci can be diversified by a xenogeneic diversification system that employs sites and mechanisms that differ significantly from those of humans. Some of the opportunities presented by the advent of rearrangement of a guest human Ig locus in the bovine B lineage are addressed by the following specific aims:AIM 1: Determine the extent and mechanism of human antibody diversification in HAC-transgenic, cloned cattle. AIM 2: Examine the synthesis, cell surface display and secretion of human immunoglobulin by HAC-transgenic bovine B-lineage cells.AIM 3: Study the humoral immune responses of cloned, HAC-transgenic calves. This study of cloned cattle containing a human artificial chromosome offers an opportunity to determine the effects of transplanting the gene assemblies responsible for human antibody production into cattle. The successful generation of significant amounts of human antibodies has the important implications for basic biology outlined above. Because cattle are large animals that have the potential to produce very large amounts of antibody, these studies could have an impact on the technology of human antibody production for use in the clinic.

已有可能构建包含整个未重排的人类重链和轻链Ig基因座的人类人工染色体(HAC)，并将其引入牛成纤维细胞。利用克隆技术的进步，已经有可能使用这些转HAC基因的成纤维细胞来产生牛胎儿和小牛。这些克隆的转基因牛将为一个项目提供实验材料，该项目将确定HAc携带的人Ig基因座在牛细胞环境中进行重排、Ig表达和产生一级抗体的能力。虽然我们假设在这些动物中将形成不同的人类抗体谱系，但牛B血统环境是否会支持产生不同的人类抗体谱系仍是一个悬而未决的问题。人类和牛科用来使抗体基因多样化的机制和部位有很大的不同。没有实验机构预测HAc携带的人类免疫球蛋白基因座的重排是有限的，就像牛的免疫球蛋白基因座一样，还是像人类和转基因小鼠那样广泛。这项实验将使我们能够研究物种特有的细胞生理学与整个未重排的免疫球蛋白基因座序列的相互作用，以确定免疫球蛋白基因多样化的机制、程度和位置。转人重链基因和轻链基因的牛的出现，使得检测来自一个物种的未重排免疫球蛋白基因座与另一个物种的多样性系统的兼容性成为可能。虽然这个问题已经在使用相同机制和保留多样性的物种之间的遗传嵌合体(即“人到鼠”)中进行了探索，但在那些不使用相同机制和多样性的物种中还没有研究过这个问题。人到牛Ig基因座嵌合体的创建提供了一个机会，以确定人类Ig基因座可以在多大程度上通过异种多样化系统实现多样化，该系统采用与人类显著不同的位点和机制。牛B血统中客体人类免疫球蛋白基因重排带来的一些机会被以下特定目标所解决：目的1：确定HAC转基因克隆牛中人类抗体多样化的程度和机制。目的2：检测转HAC基因牛B系细胞合成、细胞表面展示和分泌人免疫球蛋白的情况。目的3：研究克隆、转HAC基因小牛的体液免疫应答。这项对含有人类人工染色体的克隆牛的研究提供了一个机会，来确定将负责产生人类抗体的基因组件移植到牛身上的效果。大量人类抗体的成功产生对上述基础生物学具有重要意义。由于牛是有可能产生大量抗体的大型动物，这些研究可能会对临床使用的人类抗体生产技术产生影响。