GFP Reporters for the chondrocyte lineage

描述（由申请人提供）：通过传统光学显微镜技术表征的对受损关节软骨的细胞修复反应表明最初形成生长板软骨的相同软骨细胞谱系的再活化。然而，用现有技术鉴定成人软骨内谱系的细胞组分是不可能的，也不可能检验关节软骨内的细胞来源于软骨细胞谱系的明显不同的肢体的假设。该提案旨在引入一套新的遗传标记工具，用于形成关节软骨和生长板软骨的软骨细胞的谱系研究。基于我们先前构建和验证启动子-GFP报告基因转基因小鼠用于研究骨祖细胞谱系的经验，提出了用于软骨细胞谱系的类似策略。该计划整合了来自四个不同研究者的输入，并结合了重组工程和BAC转基因，早期肢体发育生物学和活细胞和固定组织切片中的GFP荧光成像的分子技能。从早期肢体发育研究中选择了驱动GFP的遗传单位作为可能的候选者，以识别处于确定分化阶段的细胞。已经选择了用于快速生产BAC-GFP构建体的经证实的方案。用于快速筛选从现有集落获得或内部生产的BAC转基因小鼠的方法将鉴定遗传单位，所述遗传单位可以单独或组合用于鉴定可确定的软骨细胞分化水平。这项技术驱动的赠款将测试的假设是，关节软骨的软骨细胞与形成生长板的软骨细胞具有明显不同的分化途径。具体地，关节软骨细胞从发育中关节的区间细胞发育，其最终产生滑膜关节的许多结构。根据这一推理，将测试生长板软骨细胞的遗传单位将包括：发育肢体中的间充质软骨形成祖细胞，FGF 10;前软骨细胞（前软骨缩合），Sox 9和Col 1a 1;增殖软骨细胞，Sox 9和Col 2a 1;前肥大软骨细胞，IHH;肥大软骨细胞，Col 10a 1。用于鉴定关节谱系细胞的遗传单位将包括：前区间细胞，Dlx 5和Col 2al;区间细胞，Gdf 5;早期关节软骨细胞，Pg 4（润滑素）和成熟关节细胞，tnc（腱生蛋白c）和Col 2al。从该提议中产生的用于谱系鉴定的经验证的遗传单位将可用于软骨细胞群落，并将形成用于命运定位、定向和诱导表达、多重化和载体递送的更先进表达构建体的基础。 公共卫生相关性：报告软骨细胞谱系内分化水平的视觉标记将是研究关节软骨疾病的重要进展。了解从祖细胞到完全分化的细胞的相对分布，特别是如果关节软骨和生长板软骨来自不同的分支，将对细胞发病机制提供基本的了解，并指出可能以积极的方向改变谱系进展的药物。然而，GFP报告基因的最大应用可能是在再生医学中，其中来自供体的祖细胞（用一种报告基因颜色标记）被植入受损宿主的关节软骨（用另一种颜色标记）。这种方法将表明供体成功分化为适当的细胞类型并与宿主关节软骨细胞整合。目前的标记物不能进行这种区分，这种区分限制了可以定向形成关节软骨的软骨细胞祖细胞的发育进展。该基金将产生的GFP工具集将是关节疾病再生医学更基于知识的第一步。

DESCRIPTION (provided by applicant): The cellular repair response to damaged articular cartilage that has been characterized by traditional light microscopic technology suggests reactivation of the same chondrocyte lineage that initially formed the growth plate cartilage. However identifying the cellular components of the lineage within adult cartilage is not possible with available technology, nor is it possible to test the hypothesis that cells within the articular cartilage are derived from a distinctly different limb of the chondrocyte lineage. This proposal is designed to introduce a new tool set of genetic markers for lineage studies in the chondrocytes that form the articular and growth plate cartilage. Based on our prior experience of building and validating promoter-GFP reporter transgenic mice for studying the osteoprogenitor lineage, a similar strategy is proposed for the chondrocyte lineage. The plan integrates input from four different investigators and combines the molecular skills of recombineering and BAC transgenesis, early limb developmental biology and GFP fluorescence imaging in living cell and fixed histological section. The genetic units for driving GFP have been chosen from early limb development studies as likely candidates to identify cells at defined stages of differentiation. A proven protocol for the rapid production of BAC-GFP constructs has been selected. The methods for the rapid screening of BAC transgenic mice, obtained from existing colonies or produced in house, will identify genetic units which can either alone or in combination be used to identify a definable level of chondrocyte differentiation. The hypothesis that this technology driven grant will test is that the chondrocytes of the articular cartilage have a distinctly different pathway of differentiation than the chondrocytes that form the growth plate. Specifically, articular chondrocytes develop from the interzone cells of the developing joint which ultimately give rise to many of the structures of the synovial joint. Following this reasoning, the genetic unit that will be tested for the growth plate chondrocytes will include: mesenchymal chondrogenic progenitor cells in the developing limb, FGF10; prechondrocytes (precartilage condensation), Sox9 and Col1a1; proliferating chondrocytes, Sox9 and Col2a1; prehypertrophic chondrocytes, IHH; hypertrophic chondrocytes, Col10a1. The genetic units to identify cell of the articular lineage will include: pre-interzone cells, Dlx5 and Col2a1; interzone cells, Gdf5; early articular chondrocytes, Pg4 (lubricin) and mature articular cells, tnc (tenascin c) and Col2a1. The validated genetic units for lineage identification produced from this proposal will be made available to the chondrocyte community and will form the basis for more advanced expression constructs for fate mapping, directed and inducible expression, multiplexing and vector delivery. PUBLIC HEALTH RELEVANCE: Visual markers that report the level of differentiation within the chondrocyte lineage will be a significant advance for studying diseases of articular cartilage. Knowing the relative distribution of cells from progenitor to fully differentiated, particularly if articular and growth plate cartilage arise from a different branch, will give fundamental insight into cellular pathogenesis and point to agents which might alter lineage progression in a positive direction. However the greatest application of the GFP reporters may be in regenerative medicine in which progenitor cells from a donor (marked with one reporter color) are implanted into the articular cartilage of an injured host (marked with another color). This approach will indicate the success of the donor to differentiate to the appropriate cell type and integrate with the host articular chondrocytes. Current markers cannot make this distinction that has restrained progress in developing chondrocyte progenitors that can be directed to form articular cartilage. The GFP tool set that this grant will produce will be the first step in a more knowledge based approach to regenerative medicine for diseases of the joint.