Center for Musculoskeletal Disease Research, Departments of Internal Medicine and Orthopaedic Surgery, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Center for Musculoskeletal Disease Research, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Cartilage. 2021 Dec;13(2_suppl):1720S-1733S. doi: 10.1177/19476035211057243. Epub 2021 Nov 22.
To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity.
A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis.
Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification.
This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.
为了支持针对软骨生成的治疗方法的临床前评估，我们的目标是生成一种能够非侵入性地报告内源性软骨生成活性的大鼠品系。
构建了一个转基因，其中双荧光素酶（萤火虫荧光素酶）和荧光（mCherry）报告基因的表达受大鼠 Col2a1 调控序列的控制。候选系在 Lewis 背景下建立，并通过连续生物发光成像以及在几种组织中测量分子报告基因水平来进行特征描述。在原位和异位软骨生成模型中评估了报告基因品系的敏感性和特异性。
从软骨区域（包括附肢滑膜关节、脊柱、胸骨、鼻子和耳廓）中检测到大量的生物发光信号。1 月龄时生物发光辐射强度很高，随着发育的继续迅速下降，但在 2 岁的动物中仍可检测到。外植体成像和免疫组织化学证实，两种分子报告基因均定位于软骨。将野生型骨髓基质细胞植入年轻成年和老年报告大鼠的骨软骨缺损中，导致关节内报告基因活性随软骨组织修复而呈时间依赖性升高。为了刺激异位、软骨内骨形成，骨形态发生蛋白 2 在比目鱼肌中过表达，导致与异位骨化密切相关的生物发光信号。
该品系可帮助开发刺激软骨修复和软骨内骨形成或抑制与异位骨化相关的软骨生成的策略。
