Abstract
Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens.
Highlights
Monoclonal antibodies are an important pillar in the treatment of multiple disorders such as cancer, inflammatory diseases, and orphan diseases [1,2,3]
By using a stop-codon reversion assay we show that IglhuVK, IghhuVH DT40 cells diversified the functional human heavy and light chain genes by gene conversion, suggesting that these transgenes, when inserted into fully transgenic chickens, will create a diverse repertoire of human antibodies in B cells in vivo
To generate cell lines with a single knockout of either immunoglobulin light (Igl) or Igh, the selectable markers consisted of a puromycin resistance gene and an enhanced green fluorescent protein (eGFP) gene
Summary
Monoclonal antibodies (mAB) are an important pillar in the treatment of multiple disorders such as cancer, inflammatory diseases, and orphan diseases [1,2,3]. With the development of hybridoma technology, it became possible to produce mAB in mice [4]. Because of their murine origin, these antibodies are immunogenic in humans [5,6]. Chimeric antibodies, humanized antibodies and fully human antibodies from phage display libraries were created using recombinant DNA techniques [7,8,9,10] Another attempt to solve this problem was to create transgenic animals carrying human immunoglobulin loci in order to produce human sequence antibodies directly without further manipulation [11,12,13,14,15]. The complex genetic modifications necessary to produce human antibodies in chickens (knockout of endogenous immunoglobulins and insertion of human transgenes) can be accomplished in cultured primordial germ cells, leading to the creation of fully transgenic birds. [16,17]
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