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

已经有可能构建含有整个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。 利用克隆技术的进步，已经有可能使用这些转基因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，该项目将确定在牛细胞的环境中，携带的人类IG基因座进行重排、IG表达和产生一级抗体库的能力。 虽然我们假设在这些动物中将形成人抗体的多样性库，但牛B谱系环境是否将支持人抗体的多样性库的产生是一个悬而未决的问题。 人和牛科动物使抗体基因多样化的机制和位点存在重大差异。 目前还没有任何实验能够预测，人类携带的人类IG基因座的重排是否会像牛的IG基因座那样是有限的，或者像人类和转基因小鼠那样是广泛的。 这个实验将使我们能够调查的相互作用的物种特异性细胞生理与整个未重排的免疫球蛋白基因座的序列，在确定免疫球蛋白基因多样化的机制，程度和网站。 完整人重链基因座和完整人轻链基因座的转基因牛的可用性使得可以检查来自一个物种的未重排IG基因座与另一物种的多样化系统的相容性。 虽然这个问题已经在使用相同机制和库多样化位点的物种之间的遗传嵌合体（即“人对小鼠”）中进行了探索，但尚未在那些不使用的物种中进行研究。 “人-牛”IG基因座嵌合体的产生提供了确定人IG基因座可以通过异种多样化系统多样化的程度的机会，所述异种多样化系统采用与人显著不同的位点和机制。 牛B谱系中的客体人IG基因座重排的出现所带来的一些机会通过以下具体目标来解决：目的1：确定人抗体多样化的程度和机制在TAC转基因克隆牛中。 目标2：目的3：研究转BAC基因牛B系细胞的体液免疫应答。 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，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。 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。 由于牛是大型动物，具有产生大量抗体的潜力，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。

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.