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

构建包含完整的未重排的人重链和轻链Ig基因座的人人工染色体（HAC）并将其导入牛成纤维细胞已成为可能。利用先进的克隆技术，已经有可能使用这些转基因hac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、表达和产生一抗库的能力。虽然我们假设这些动物体内会形成多种人类抗体，但牛B系环境是否会支持产生多种人类抗体仍是一个悬而未决的问题。人类和牛科动物分化抗体基因的机制和位点存在重大差异。目前还没有大量的实验可以预测hac携带的人类Ig基因座的重排是有限的，就像牛的一样，还是广泛的，就像在人类和转基因小鼠中看到的那样。该实验将使我们能够研究物种特异性细胞生理与免疫球蛋白全未重排位点序列的相互作用，从而确定免疫球蛋白基因多样化的机制、程度和位置。人类重链基因座和轻链基因座的牛转基因的可获得性，使得检测一个物种未重排的Ig基因座与另一个物种多样化系统的相容性成为可能。尽管这个问题已经在基因嵌合体(如：在使用相同机制和种群多样化位点的物种之间（“人到老鼠”），尚未对那些不使用相同机制和位点的物种进行研究。“人-牛”Ig基因座嵌合体的创建提供了一个机会，可以确定人类Ig基因座通过异种多样化系统可以多样化的程度，该系统使用的位点和机制与人类有很大不同。在牛B系中出现的客人人Ig位点重排所带来的一些机会可以通过以下具体目的来解决：目的1：确定人抗体多样化的程度和机制在转hac的克隆牛中。目的2：用转hac的牛b系细胞检测人免疫球蛋白的合成、细胞表面显示和分泌。目的3：研究克隆的、转hac的小牛的体液免疫反应。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，以确定将负责人类抗体产生的基因组装体移植到牛体内的效果。成功产生大量的人类抗体对上述基础生物学具有重要意义。由于牛是大型动物，有可能产生大量抗体，这些研究可能对临床使用的人类抗体生产技术产生影响。

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.