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Engineering and validation of two conditional multi-gene mouse models of skeletal development

两种条件多基因小鼠骨骼发育模型的工程和验证

基本信息

批准号：
10043332
负责人：
Kimberly Lynn Cooper
金额：
$ 19.68万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-13 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10043332
关键词：
AQP9 gene Aging Alleles Animal Model Animals Breeding CRISPR/Cas technology Cartilage Cell Death Cell Membrane Permeability Cell membrane Cells Chondrocytes Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Complex Complex Genetic Trait Custom DNA Development Diffuse Digit structure Disease Drosophila genus Elements Embryo Engineering Enterobacteria phage P1 Cre recombinase Epiphysial cartilage Face Frequencies Future Gene Proteins Genes Genetic Genetic Recombination Genotype Glycerol Growth Guide RNA Guidelines Human Hypertrophy Investments Ion Channel Joints Knock-out Knockout Mice Laboratory mice Ligand Binding Ligands Limb structure Liquid substance Location Loss of Heterozygosity LoxP-flanked allele Maintenance Mediating Mesenchyme Modeling Monophenol Monooxygenase Mosaicism Mus Musculoskeletal Development Musculoskeletal Diseases Mutagenesis Mutation Osteoblasts Outcome Pathway interactions Pattern Phenotype Physical condensation Process Proteins RNA Polymerase III Reporting Reproducibility Research Ribonucleases Rice Risk Role Signal Pathway Signal Transduction Site Skeletal Development Skeletal system Skeleton Swelling System Testing Time Tissues Transcript Transfer RNA Transforming Growth Factor beta Transgenes Transgenic Mice Transgenic Organisms U6 small nuclear RNA Urea Validation Water Work Zebrafish aquaporin 3 articular cartilage base conditional knockout cost endonuclease innovation loss of function loss of function mutation member model development mouse model new technology null mutation offspring programs promoter protein expression receptor recruit skeletal skeletal disorder solute tool uptake water channel zygote

项目摘要

A deep understanding of the genetic mechanisms of musculoskeletal development and disease requires study of genes that are frequently pleiotropic and/or highly redundant. Currently, the common strategy to study such complex genetic traits in mice is to combine multiple homozygous conditional (floxed) alleles with a tissue-specific and/or temporally inducible Cre recombinase transgene. However, such breeding strategies to understand three or more loci require an extraordinary investment of money, time, and mice to obtain just a few animals of the desired genotype, often limiting enquiry to a particular tissue and developmental stage. Here, we propose an innovative approach in mice using just three hemizygous transgenes to conditionally induce multi-gene, bi-allelic, loss-of-function mutations to study complex genetic control of development and maintenance of the limb skeleton. We previously demonstrated highly efficient CRISPR/Cas9 mutagenesis from genetically encoded elements that included the Rosa26-LoxStopLox-Cas9 transgene. Crossing these mice with a tissue-specific and/or temporally-inducible Cre transgenic line provides an opportunity to control the timing and/or location of CRISPR/Cas9 activity. The third transgene required for this proposed system is a `polycistronic tRNA-gRNA' (PTG) array of CRISPR guide RNAs interspersed with tRNA sequences. These transcripts recruit endogenous RNases to cleave apart gRNAs, and processed gRNAs can complex with Cas9 protein to induce sequence-targeted double strand breaks in DNA. PTG arrays have been effective in rice, Drosophila, zebrafish, and cultured human cells, but they remain untested in mice. Using this strategy, we plan to engineer and validate two models of skeletal development. In Aim 1, we will engineer a Smad1, Smad5, and Smad8 conditional knockout strategy to target loss of BMP/GDF signaling at a critical bottleneck. This will be a valuable model to thoroughly assess pathway function during limb patterning, digit condensation and interdigital cell death, chondrocyte specification and maintenance, and osteoblast specification and maintenance. In Aim 2, we will engineer an aquaglyceroporin (AQP3, AQP7, and AQP9) conditional knockout strategy. This model will allow us to test the hypothesis that these membrane channels that increase plasma membrane permeability to water and small uncharged osmolytes are necessary for growth plate hypertrophic chondrocyte swelling and maintenance of articular cartilage. Since the purpose of this R21 proposal is the engineering and validation of new technology and animal models, our primary emphasis will be to determine the efficiency and reproducibility of such strategies and to establish a set of guidelines for future implementation. Successful outcomes of this work will provide a wealth of opportunities to understand the importance of these two complex genetic systems, and the strategy has broad potential to accelerate research and reduce the costs associated with mouse models of development and disease.

深入了解肌肉骨骼发育和疾病的遗传机制需要研究经常是多效性和/或高度冗余的基因。目前，共同战略在小鼠中研究这种复杂的遗传性状是将联合收割机多个纯合条件（floxed）等位基因与组织特异性和/或时间诱导型Cre重组酶转基因。然而，这种育种策略，我知道三个或更多的基因座需要金钱、时间和鼠标的非凡投资，才能获得一个基因座。很少有动物具有所需的基因型，通常将调查限制在特定的组织和发育阶段。在这里，我们提出了一种在小鼠中仅使用三种半合子转基因的创新方法，有条件地诱导多基因、双等位基因、功能缺失突变，以研究复杂的遗传控制，四肢骨骼的发育和维持。我们之前展示了高效的CRISPR/Cas9 在一些实施方案中，本发明涉及从包括Rosa 26-LoxStopLox-Cas9转基因的遗传编码元件进行诱变。将这些小鼠与组织特异性和/或时间诱导型Cre转基因系杂交，提供了一种新的方法。这是控制CRISPR/Cas9活性的时间和/或位置的机会。第三个转基因需要这个所提出的系统是CRISPR引导RNA的“多顺反子tRNA-gRNA”（PTG）阵列，序列的这些转录物募集内源性RNA酶以切割分开gRNA，并且加工的gRNA可以与Cas9蛋白复合以诱导DNA中的序列靶向双链断裂。PTG阵列已被在水稻、果蝇、斑马鱼和培养的人类细胞中有效，但它们尚未在小鼠中进行测试。使用这种策略，我们计划工程师和验证骨骼发育的两个模型。目标1：将设计Smad 1、Smad 5和Smad 8条件性敲除策略，以靶向BMP/GDF信号传导的丢失在一个关键的瓶颈。这将是一个有价值的模型，以彻底评估通路功能，在肢体图案化，手指压缩和趾间细胞死亡，软骨细胞特化和维持，以及成骨细胞的规格和维护。在目标2中，我们将设计一种水甘油孔蛋白（AQP 3，AQP 7， AQP 9）条件性敲除策略。这个模型将使我们能够测试这些膜的假设增加质膜对水和小的不带电荷的渗透剂的渗透性的通道是必需的用于生长板肥大软骨细胞肿胀和维持关节软骨。由于目的这项R21提案是新技术和动物模型的工程和验证，我们的主要任务是重点将是确定这些策略的效率和可重复性，并建立一套今后实施的指导方针。这项工作的成功结果将提供大量机会，了解这两个复杂的遗传系统的重要性，该战略具有广泛的潜力，加速研究并降低与小鼠发育和疾病模型相关的成本。