Ovalbumin locus BACs for expression of biotherapeutics

描述（由申请人提供）：鸡卵清蛋白基因座具有每个鸡蛋两克蛋白的日生产能力，具有将大量生物药物蛋白合成到转基因母鸡的鸡蛋中的潜力。该一期SBIR项目将测试基于完整的200个酶鸡卵清蛋白基因位点使用大表达构建体的可行性，以驱动包括人源化单克隆抗体在内的生物药物蛋白质的有效表达。该计划的长期目标是在第二阶段使用这些构建体创建转基因母鸡，其将在白色中产生mg/ml浓度的生物药物蛋白。在第一阶段的建议，我们计划创建一个完整的200个酶卵清蛋白基因从两个重叠的细菌人工染色体（BAC），利用recA辅助限制性内切酶（RARE）切割技术。使用类似的技术，我们将把报告基因和生物制药基因插入卵清蛋白基因转录区域内的各个位置。最后，我们将使用体外和体内测定来测定来自这些构建体的蛋白质表达。

DESCRIPTION (provided by applicant): With a daily production capacity of two grams of protein per egg, the chicken ovalbumin gene locus has the potential to synthesize large amounts of biopharmaceutical proteins into the eggs of transgenic hens. This phase I SBIR project will test the feasibility of using large expression constructs, based on the complete 200 kilobase chicken ovalbumin gene locus, to drive efficient expression of biopharmaceutical proteins including humanized monoclonal antibodies. The longer-term objectives of the program are to use these constructs in the creation of transgenic hens, in phase II that will produce biopharmaceutical proteins at mg/ml concentration in egg white. In this phase I proposal, we plan to create a complete 200 kilobase ovalbumin gene from two overlapping bacterial artificial chromosomes (BAC) by utilizing recA-assisted restriction endonuclease (RARE) cleavage techniques. Using similar techniques, we will then insert reporter and biopharmaceutical genes into various locations within the transcribed region of the ovalbumin gene. Finally, we will assay protein expression from these constructs using in vitro and in vivo assays.