Engineering Large Chromosomal Deletions in Mice to Advance Precision Oncology

项目摘要/摘要 基因组缺失，包括局部和大臂水平的染色体缺失，都是常见的遗传事件 促进癌症发生和发展的物质。焦点删除已经对机制产生了重要的见解 通过帮助识别位于缺失基因座的肿瘤抑制基因(TSG)，揭示肿瘤发生的潜在机制。在……里面 相比之下，人们对癌症中大量染色体缺失的生物学和表型影响知之甚少。 尽管它们的发生频率是焦点删除的30倍。我们和其他研究人员已经证明 这样的删除影响了多个相邻的临时政府的活动，并构成了 肿瘤发生学。此外，由大规模缺失引发的多个基因的随之而来的丢失可能导致 与失去单个TSG所产生的表型根本不同的表型，并可能提供 出乎意料的治疗机会。因此，这些丢失应该被认为是不同的遗传事件 他们的分析应该集中在染色体缺失的综合影响上，而不是 一个关键的TSG。基因工程小鼠(GEM)模型为研究 肿瘤生物学中遗传变异的后果。然而，由于靶向的克隆能力有限， 载体(~300kb)与传统基因打靶中靶上同源重组事件的稀有性 在小鼠身上模拟大的染色体缺失的技术已经被证明具有高度的挑战性，并且在体内的作用 因此，对这类损伤在肿瘤发生中的作用研究严重不足。为了填补这一关键缺口，我们有 开发了一种在大遗传距离上进行染色体工程的创新方法 CRISPR/Cas9技术在小鼠胚胎干细胞中的应用。生成的ES克隆携带所需的 基因组编辑使创造准确模仿癌症相关的条件性基因敲除小鼠成为可能 大量删除。考虑到前列腺癌中大量染色体缺失的发生率和重要性，这里 我们建议利用我们的创新方法来生成并充分描述两个新的宝石模型， 每个都有一个共同但不同的前列腺癌相关的大片段缺失，目的是验证和 认为这些模型在遗传学和生物学上都是人类前列腺癌的可靠代表。在……里面 目标1，我们将使用CRISPR/Cas9技术开发有条件的小鼠品系，以针对常见的缺失大鼠 人类前列腺癌的染色体定位。 在目标2中，我们将建立携带前列腺癌的小鼠模型 大量染色体缺失以分析它们对前列腺癌发生的生物学和表型影响。 综上所述，这些模型及其特征将满足此次赠款机会的多个目标。 成功完成这些调查不仅将极大地有助于实施精确度 肿瘤学研究也提高了动物模型对转化性研究的适用性。

Project Summary/Abstract Genomic deletions, including both focal and large arm-level chromosomal deletions, are frequent genetic events that promote cancer initiation and progression. Focal deletions have yielded significant insights into mechanisms underlying tumorigenesis by helping identify tumor suppressor genes (TSGs) residing in the deleted loci. In contrast, little is known about the biological and phenotypic impacts of large chromosomal deletions in cancer despite their occurrence 30 times more frequently than focal deletions. We and other researchers have shown that such deletions impact the activities of multiple neighboring TSGs and constitute a driving force in tumorigenesis. Additionally, concomitant loss of multiple genes triggered by broad-scale deletions can create phenotypes that differ fundamentally from those arising from the loss of a single TSG, and may offer unanticipated therapeutic opportunities. These losses should therefore be considered as distinct genetic events and their analysis should be focused on the integrated effects of chromosomal deletion rather than the impact of a single critical TSG. Genetically engineered mouse (GEM) models provide an ideal tool for investigating the consequences of genetic aberrations in tumor biology. However, due to the limited cloning capacity of targeting vectors (~300 Kb) and the rarity of on-target homologous recombination events in traditional gene-targeting technology, modelling large chromosomal deletions in mice has proven highly challenging, and the in vivo role of such lesions in tumorigenesis has therefore been significantly understudied. To fill this critical gap, we have developed an innovative approach to chromosomal engineering over large genetic distances through CRISPR/Cas9 technology in mouse embryonic stem (ES) cells. The resulting ES clones carrying the desired genome edits make possible the creation of conditional knockout mice that accurately mimic cancer-associated large deletions. Given the prevalence and significance of large chromosomal deletions in prostate cancer, here we propose to utilize our innovative approach to generate and fully characterize two novel GEM models that each harbor a common but distinct prostate cancer-associated large deletion, with a goal of validating and credentialing these models as genetically and biologically robust representations of human prostate cancer. In Aim 1, we will develop conditional mouse lines using CRISPR/Cas9 technology to target commonly deleted large chromosomal loci in human prostate cancer. In Aim 2, we will develop mouse models of prostate cancer harboring large chromosomal deletions to analyze their biological and phenotypic impacts on prostate cancer development. Taken together, these models and their characterization will meet multiple goals of this grant opportunity. Successful completion of these investigations will not only contribute greatly to the implementation of precision oncology research but also enhance the applicability of animal models to translational research.