A Transgenic Rat for Noninvasive Assessment of Chondrogenic Activity in vivo

 描述(由申请人提供)：关节损伤的啮齿动物模型对于开发下一代治疗策略以预防创伤后骨关节炎(PTOA)至关重要。近几十年来，基因工程技术应用于特征明确的小鼠基因组，导致了转基因和基因敲除品系的革命性发展。虽然这些小鼠模型现在是骨科研究的基本工具，但类似的大鼠模型一直落后。然而，随着对大鼠基因组知识的不断提高，再加上基因组编辑的不断进步，现在转基因大鼠的开发变得更加实用。大鼠和小鼠之间有重要的差异，这使得后一种物种更适合在体内进行新疗法的初步测试。例如，RA通常用于评估基于干细胞的新策略，以刺激骨软骨组织修复，而小鼠关节太小，不能用于此目的。通过对基因工程干细胞进行非侵入性成像以表达分子记者的能力，大鼠的损伤模型已经得到了改进。在现有的报道中，荧光素酶催化强烈的生物发光反应，可以用现有的成像平台进行定量测量。如果对大鼠进行基因工程，使其在致力于软骨形成系的细胞中特异性地表达荧光素酶，这将允许对内源性软骨前体细胞(CPC)分化进行非侵入性评估。软骨和半月板缺损处的CPC软骨形成是修复的一个重要瓶颈，该大鼠模型将有助于测试克服这一瓶颈的策略。第一个项目的目标是产生一只转基因大鼠，在其中，萤火虫荧光素酶(Fluc)和LacZ由致力于软骨细胞系的细胞特异性表达，而Renilla荧光素酶(RLuc)和可诱导的CreERT2重组酶由所有细胞组成地表达。利用软骨细胞中II型胶原表达的后天特异性，我们将使用Col2a1基因的调控序列来控制flc和lacZ的表达。一旦双荧光素酶转基因大鼠被制造出来，第二个项目的目标将是用两个关节损伤的活体模型来证明它的实用性：(I)在野生型大鼠中注射转基因CPC造成骨软骨缺损；(Ii)在野生型大鼠中建立PTOA的半月板切除模型，注射转基因CPC。在这些初步研究中，将对来自骨髓和滑膜的间充质干细胞进行比较。一旦转基因大鼠的特征被确定，它将作为骨科研究社区的资源提供。当与现有的生物发光分析和成像平台相结合时，该菌株将能够灵敏、定量地测量促软骨生成活性，不仅可以研究关节损伤，还可以研究软骨内骨形成(例如，骨折愈合)。这个 将他莫昔芬诱导的CreERT2基因工程引入这只大鼠，将使其能够与未来的loxp大鼠杂交，进行功能得失研究，极大地扩大其应用范围，并最终加速骨科研究。

 DESCRIPTION (provided by applicant): Rodent models of joint injury are vital for the development of next-generation treatment strategies to prevent post-traumatic osteoarthritis (PTOA). In recent decades, the application of genetic engineering techniques to the well-characterized genome of mice has resulted in the revolutionary development of transgenic and knockout strains. While these mouse models are now a fundamental tool in orthopaedic research, similar rat models have been lagging. However, an improving knowledge of the rat genome combined with continued advances in genome editing now make transgenic rat development more practical. There are important differences between rats and mice that make the latter species preferred for initial testing of novel therapeutics in vivo. For example, the ra is routinely used for evaluating new stem cell-based strategies to stimulate osteochondral tissue repair, whereas mouse joints are too small for this purpose. Rat models of injury have been advanced by the ability to noninvasively image stem cells genetically engineered to express molecular reporters. Among the available reporters, the luciferases catalyze intense bioluminescent reactions that can be measured quantitatively with established imaging platforms. If rats were genetically engineered to express a luciferase specifically within cells committed to the chondrogenic lineage, this would allow noninvasive evaluation of endogenous chondroprogenitor cell (CPC) differentiation. CPC chondrogenesis within cartilage and meniscal defects is an important bottleneck to repair, and this rat model would help test strategies to overcome this bottleneck. The first project aim will be to generate a transgenic rat in which Firefly luciferase (FLuc) and LacZ are expressed specifically by cells committed to the chondrogenic lineage, while Renilla luciferase (RLuc) and inducible CreERT2 recombinase are expressed constitutively by all cells. Exploiting the postnatal specificity of type II collagen expression within cells of the chondrogenic lineage, we will use regulatory sequences from the Col2a1 gene for controlling FLuc and LacZ expression. Once the dual- lucifase trasngenic rat has been made, the second project aim will be to demonstrate its utility using two in vivo models of joint injury: (i) osteochondral defects made in wild type rats receiving transgenic CPCs, and (ii) a meniscectomy model of PTOA in wild type rats, injecting transgenic CPCs. For these initial studies, mesenchymal stem cells from bone marrow and synovium will be compared. Once the transgenic rat has been characterized, it will be made available as a resource to the orthopaedic research community. When combined with existing bioluminescence assays and imaging platforms, this strain will permit sensitive, quantitative measuring of pro-chondrogenic activity i studies of not only joint injury but also endochondral bone formation (e.g., fracture healing). The engineering of tamoxifen-inducible CreERT2 into this rat will allow it to be crossed with future LoxP rats for gain and loss of function studies, greatly broadening its application and, ultimately accelerating orthopaedic research.