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

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 要研究小鼠的这种复杂的遗传特征，就需要将多个纯合的条件(花)等位基因与 组织特异性和/或时间可诱导的Cre重组酶转基因。然而，这种繁殖策略是为了 了解三个或更多的基因座需要投入大量的金钱、时间和鼠标，才能获得 很少有动物具有所需的基因型别，通常仅限于特定的组织和发育阶段。 在这里，我们提出了一种创新的方法，在小鼠身上只使用三个半合子转基因来 有条件地诱导多基因、双等位基因、功能丧失突变以研究复杂的遗传控制 四肢骨骼的开发和维护。我们之前展示了高效的CRISPR/CAS9 从包括rosa26-LoxStopLox-Cas9转基因的遗传编码元素进行突变。 将这些小鼠与组织特异性和/或临时诱导的Cre转基因系杂交，可以提供 有机会控制CRISPR/CAS9活动的时间和/或地点。为此所需的第三种转基因 所提出的系统是散布着tRNA的CRISPR引导RNA的‘多顺反子tRNA-gRNA’(PTG)阵列 序列。这些转录本招募内源RNA酶来裂解gRNA，而处理后的gRNA可以 与Cas9蛋白的复合体诱导DNA序列靶向双链断裂。PTG阵列已经被 在大米、果蝇、斑马鱼和培养的人类细胞中有效，但它们仍未在老鼠身上进行测试。 使用这一策略，我们计划设计和验证两种骨骼发育模型。在目标1中，我们 将设计Smad1、Smad5和Smad8条件敲除策略，以针对BMP/GDF信号的丢失 在一个关键的瓶颈。这将是一个有价值的模型，以彻底评估路径功能在四肢 图案化、指间凝缩和指间细胞死亡、软骨细胞规格和维护； 成骨细胞的规范和维护。在目标2中，我们将设计一种水甘油孔蛋白(AQP3、AQP7和 AQP9)条件淘汰赛策略。这个模型将允许我们测试这些膜的假设 增加质膜对水的通透性的通道和小的不带电荷的渗透压是必要的 适用于生长板肥大的软骨细胞肿胀和关节软骨的维护。由于……的目的 R21计划是新技术和动物模型的设计和验证，我们的主要 重点将是确定这些战略的效率和可重复性，并建立一套 今后实施的指导方针。这项工作的成功成果将提供丰富的机会 了解这两个复杂遗传系统的重要性，该战略具有广泛的潜力 加快研究并降低与小鼠发展和疾病模型相关的成本。

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.