A Transgenic Rat for Noninvasive Assessment of Chondrogenic Activity in vivo

 描述（由申请方提供）：啮齿动物关节损伤模型对于开发预防创伤后骨关节炎（PTOA）的下一代治疗策略至关重要。近几十年来，基因工程技术在小鼠基因组中的应用，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很落后。然而，对大鼠基因组的不断了解加上基因组编辑的不断进步，现在使转基因大鼠的开发更加实用。大鼠和小鼠之间存在重要差异，这使得后一种物种优选用于体内新疗法的初始测试。例如，ra通常被用于评估新的基于干细胞的刺激骨软骨组织修复的策略，而小鼠关节太小，无法达到此目的。大鼠损伤模型已经通过非侵入性成像干细胞基因工程表达分子报告的能力而得到了发展。在可用的报告基因中，荧光素酶催化强烈的生物发光反应，可以用已建立的成像平台定量测量。如果对大鼠进行基因工程改造，使其在软骨形成谱系的细胞内特异性表达荧光素酶，这将允许对内源性软骨祖细胞（CPC）分化进行无创评估。软骨和骨缺损中的CPC软骨形成是修复的重要瓶颈，这种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种转基因大鼠，其中萤火虫荧光素酶（FLuc）和LacZ由致力于软骨形成谱系的细胞特异性表达，而海肾荧光素酶（RLuc）和诱导型CreERT2重组酶由所有细胞组成型表达。利用软骨细胞系中II型胶原蛋白表达的出生后特异性，我们将使用Col2a1基因的调控序列来控制FLuc和LacZ的表达。一旦制备了双荧光素酶转基因大鼠，第二个项目的目的将是使用两种关节损伤的体内模型来证明其效用：（i）在接受转基因CPC的野生型大鼠中制备的骨软骨缺损，和（ii）注射转基因CPC的野生型大鼠中PTOA的骨切除术模型。在这些初步研究中，将比较来自骨髓和滑膜的间充质干细胞。一旦转基因大鼠被鉴定，它将作为骨科研究界的一种资源。当与现有的生物发光测定和成像平台相结合时，该菌株将允许在关节损伤和软骨内骨形成（例如，骨折愈合）。的 将他莫昔芬诱导的CreERT2工程化到这种大鼠中将允许它与未来的LoxP大鼠杂交，用于功能获得和丧失的研究，极大地拓宽了其应用，并最终加速矫形研究。

 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.