GFP Reporters for the chondrocyte lineage

软骨细胞谱系的 GFP 报告基因

• 批准号: 7673615
• 负责人: David W. Rowe
• 金额: $ 16.28万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7673615
• 关键词: Adult; Automobile Driving; Breeding; Candidate Disease Gene; Cartilage; Cells; Chondrocytes; Color; Communities; Cryoultramicrotomy; Developmental Biology; Disease; Embryo; Embryo Transfer; Epiphysial cartilage; FGF10 gene; Female; Fluorescence Microscopy; Gene Transfer Techniques; Genetic; Genetic Markers; Grant; Housing; Implant; In Vitro; Injection of therapeutic agent; Joints; Life; Light; Limb Development; Limb structure; Maps; Mesenchymal; Microscopic; Modification; Molecular; Mus; Pathogenesis; Pathway interactions; Pattern; Physical condensation; Pregnancy; Production; Proliferating; Protocols documentation; Reagent; Regenerative Medicine; Relative (related person); Reporter; Reporting; Research; Research Personnel; Screening procedure; Signal Transduction; Staging; Stem cells; Synovial joint; Techniques; Technology; Tenascin; Testing; Time; Tissues; Transgenic Mice; Transgenic Organisms; Visual; arthropathies; articular cartilage; base; cell fixing; cell type; design; expectation; experience; fluorescence imaging; in vivo; injured; insight; knowledge base; lubricin; male; mouse model; osteoprogenitor cell; progenitor; promoter; public health relevance; rapid technique; repaired; response; skills; success; tool; vector

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.

描述（由申请人提供）：以传统光学微观技术为特征的对受损的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重新激活。然而，通过可用的技术，无法识别成人软骨内部谱系的细胞成分，也无法检验假设关节软骨中的细胞源自软骨细胞谱系的截然不同的肢体。该提案旨在引入一组新的遗传标记工具，以用于构成关节和生长板软骨的软骨细胞中的谱系研究。基于我们先前在构建和验证启动子GFP报告基因谱系谱系的经验，因此提出了类似的软骨细胞谱系策略。该计划整合了四个不同研究者的输入，并结合了活细胞和固定组织学部分中重新组合和BAC转基因，早期肢体发育生物学和GFP荧光成像的分子技能。从早期的肢体发展研究中选择了用于驾驶GFP的遗传单位，因为它们可能是在定义的分化阶段鉴定细胞的候选者。已经选择了快速生产BAC-GFP构建体的验证协议。从现有菌落或内部产生的BAC转基因小鼠快速筛选的方法将识别可以单独或合并的遗传单位，以识别可确定水平的软骨细胞分化水平。该技术驱动的赠款将检验的假设是，关节软骨的软骨细胞与形成生长板的软骨细胞的分化途径截然不同。具体而言，关节软骨细胞是从发育中的关节的间区域细胞发展而来的，最终导致滑膜关节的许多结构。推理之后，将对生长板软骨细胞进行测试的遗传单位将包括：发育中的肢体中的间充质软骨祖细胞FGF10；前细胞（前冷凝），SOX9和COL1A1；增殖软骨细胞，SOX9和COL2A1；胸部内软骨细胞，IHH；肥厚的软骨细胞，COL10A1。鉴定关节谱系细胞的遗传单位将包括：互联区细胞，DLX5和COL2A1；间氮化细胞GDF5;早期关节软骨细胞PG4（润滑剂）和成熟的关节细胞，TNC（Tenascin C）和Col2A1。从本提案中产生的谱系鉴定的经过验证的遗传单位将提供给软骨细胞群落，并将为命运映射，定向和诱导表达，多重和矢量传递的更先进的表达构造构成基础。 公共卫生相关性：报告软骨细胞谱系中分化水平的视觉标记将是研究关节软骨疾病的重要进步。了解细胞从祖细胞到完全分化的相对分布，特别是如果关节和生长板软骨来自不同的分支，将使对细胞发病机理的基本见解，并指向可能在正方向上改变谱系进展的药物。但是，GFP记者的最大应用可能是在再生医学中，其中将供体的祖细胞（标有一个记者颜色标记）植入受伤宿主的关节软骨中（标有另一种颜色）。这种方法将表明捐赠者的成功与适当的细胞类型区分开，并与宿主关节软骨细胞整合。当前的标记不能使这种区别在开发软骨细胞祖细胞中限制进展，这些祖细胞可导向形成关节软骨。该赠款将产生的GFP工具设置将是基于知识的基于知识疾病的基于知识的方法的第一步。

项目成果

Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.

• DOI: 10.1002/jor.22816
• 发表时间: 2015-07
• 期刊: Journal of orthopaedic research : official publication of the Orthopaedic Research Society
• 作者: Hagiwara Y;Dyment NA;Jiang X;Jiang Ping H;Ackert-Bicknell C;Adams DJ;Rowe DW
• 通讯作者: Rowe DW