RUI: Early Guest Ig Expression and Diversity in Cloned Transgenic Cattle

这些研究将确定携带人类免疫球蛋白(Ig)基因的转基因牛的B细胞是否能产生正常的人类抗体。问题是牛重组酶(RAG-1和RAG-2)和其他牛特异性因子能否成功重排和表达人免疫球蛋白基因。其具体目的是(1)检测牛脾细胞中人免疫球蛋白的表面和可溶性形式及其mRNA的表达，(2)通过测序确定编码人轻链和重链的基因发生正常重排的程度，以及(3)确定表达的重链和轻链的谱系多样性。这些转基因研究将与血液技术公司合作完成，该公司将饲养转基因小牛。另外两个目标被提出来确定牛免疫球蛋白基因的组织。其具体目的是(4)鉴定牛重链基因座的种系元件和排列，(5)分析重排的牛基因的J-C内含子，以确定引起J-C多样性的机制(S)(模板或非模板体细胞点突变，或不同基因片段的重排)。这些研究将有助于对牛免疫球蛋白基因的基本了解。对其他脊椎动物物种的比较研究表明，这些基因的种系排列存在显著的跨物种多样性，产生多样性的机制也不同。这些研究将为这些基本机制的进化保守提供洞察力。这一结果对利用牛转基因系统表达人类特异性抗体具有重要的实际意义。该项目还将对将在阿默斯特学院参与这项研究的本科生的教育和培训做出重大贡献。

These studies will determine if B cells of transgenic cattle bearing human immunoglobulin (Ig) genes can generate normal human antibodies. The question is whether the bovine recombinases (Rag-1 and Rag-2) and other bovine-specific factors can successfully rearrange and express the human Ig genes. The specific aims are (1) to assay for expression of surface and soluble forms of human Igs and their mRNA in bovine spleen cells, (2) to determine by sequencing the extent to which the genes encoding human light and heavy chains have undergone normal rearrangements, and (3) to determine the repertoire diversity of expressed heavy chains and lambda light chain. These transgenic studies will be done in collaboration with the Hematech Corporation which will raise the transgenic fetal calves. Two additional aims are proposed to determine the organization of bovine Ig genes. The specific aims are (4) to characterize the germline elements and arrangement of the bovine heavy chain locus, and (5) to analyze the J-C introns of rearranged bovine genes to determine the mechanism(s) that give rise to J-C diversity (templated or untemplated somatic point mutations, or rearrangements of different gene segments). The studies will contribute to the basic understanding of immunoglobulin genes in cattle. Comparative studies on other vertebrate species indicate that there is significant cross-species diversity in the germline arrangement of these genes and variations in the mechanisms used to generate diversity. The studies will provide insights into the evolutionary conservation of these basic mechanisms. The results could have great practical importance for use of the bovine transgenic system to express specific human antibodies. The project will also contribute significantly to the education and training of undergraduate students who will participate in this research at Amherst College.