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

Amherst College, Amherst MA

Investigators

Abstract

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.