Engineering transgenic silkworms to produce spider silk fibers

描述(申请人提供)：真丝纤维具有许多当前和未来的生物医学应用。它们被广泛用作精细缝合材料，甚至需要更细的纤维用于眼科、神经和整容手术。丝素纤维也有望成为人工韧带和肌腱的材料，它们还有许多其他潜在的生物医学应用。许多不同的重组蛋白生产系统已经被用来试图满足当前的需求，并开发更多的丝绸生物医学应用。每一种都产生了丝蛋白，但没有一种能持续地产生有用的丝纤维。因此，这项R21探索性建议的总体目的是开发一种能够生产蜘蛛丝纤维的系统。我们的基本方法将是采用家蚕作为蜘蛛丝蛋白生产的替代宿主。高效的iggyBac系统将被用来通过合成基因编码异常大的蜘蛛丝蛋白来遗传转化突变的家蚕，这些突变的家蚕不会产生天然的丝。然后将对转基因家蚕的遗传和生化特性，特别是它们产生蜘蛛丝纤维的能力进行严格的评估。我们计划的一个关键特点是，它包括将优化我们获得纤维的能力的具体措施。这些措施包括：(I)使用编码异常大的蜘蛛丝蛋白的合成基因，(Ii)使用将异源丝蛋白表达到丝腺的启动子，丝腺天生就有纺丝的能力，(Iii)使用适当的先导肽从丝腺分泌，以及(Iv)使用替代宿主，丝蛾，它非常容易遗传转化，并自然地具备纺丝纤维的能力。在这一探索性项目中，成功分离出一种能够产生蜘蛛丝纤维的转基因蚕蛾，将为未来旨在进一步开发该系统的项目奠定基础，以生产具有预定物理性能的蜘蛛丝纤维，以优化特定的生物医学应用。这将利用目前关于蜘蛛丝抗拉强度或弹性的特定多肽基序的知识。从理论上讲，这些图案可以用不同的方式组合起来，设计出强度和弹性不同的丝绸纤维。当前项目成功完成的总体可能性增加了，因为它将由一个由三名研究人员组成的团队承担，他们在该项目的三个关键领域建立了互补的计划：蜘蛛丝(Lewis)、昆虫表达系统(Jarvis)和昆虫转化(Fraser)。蚕丝纤维在当前和未来的生物医学领域有着广泛的应用。它们被广泛用作精细缝合材料，甚至需要更细的纤维用于眼科、神经和整容手术。丝素纤维也有望成为人工韧带和肌腱的材料，它们还有许多其他潜在的生物医学应用。许多不同的重组蛋白生产系统已经被用来试图满足当前的需求，并开发更多的丝绸生物医学应用。每一种都产生了丝蛋白，但没有一种能持续地产生有用的丝纤维。因此，这项R21探索性建议的总体目的是开发一种系统，能够为这些当前和未来的生物医学应用生产蜘蛛丝纤维。我们对这一探索性项目的计划是分离出一种能够产生蜘蛛丝纤维的转基因家蚕。如果该项目成功，它将为未来的项目奠定基础，这些项目旨在进一步开发该系统，以生产具有预先确定的物理性能的蜘蛛丝纤维，以优化特定的生物医学应用。这些项目将利用目前关于特定多肽主题的知识，这些主题有助于蜘蛛丝的抗张强度或弹性。

DESCRIPTION (provided by applicant): Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers. Our basic approach will be to adopt the silkworm as a surrogate host for spider silk protein production. The highly efficient piggybac system will be used to genetically transform mutant silkworms, which produce no native silk, with a synthetic gene encoding an unusually large spider silk protein. The genetic and biochemical properties of the resulting transgenic silkworms, particularly their ability to produce spider silk fibers, will then be critically assessed. A key feature of our plan is that it includes specific measures that will optimize our ability to obtain fibers. These include: (i) using a synthetic gene encoding an unusually large spider silk protein, (ii) using a promoter that targets expression of the heterologous silk proteins to the silk gland, which is naturally equipped to spin silk fibers, and (iii) using an appropriate leader peptide for secretion from the silk gland, and (iv) using a surrogate host, the silk moth, which is highly amenable to genetic transformation and naturally equipped to spin silk fibers. The successful isolation of a transgenic silkmoth that can produce spider silk fibers in this exploratory project will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. This will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks. Theoretically, these motifs can be combined in various ways to design silk fibers differing in strength and elasticity. The overall likelihood that the current project can be successfully completed is enhanced by the fact that it will be undertaken by a team of three researchers with established, complementary programs in three areas key to the project: spider silks (Lewis), insect expression systems (Jarvis), and insect transformation (Fraser). Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers for these current and future biomedical applications. Our plan for this exploratory project is to isolate a transgenic silkworm that can produce spider silk fibers. If this project is successful, it will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. These projects will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks.