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

Amherst College, Amherst MA

Investigators

Abstract

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.