GENETIC ANALYSIS OF TYPE I COLLAGEN FUNCTION

I型前α链中特定氨基酸的修饰 胶原蛋白被认为是生物化学的主要贡献者， I型胶原纤维的物理和生物力学性质 建筑，指示矿化和 非矿化组织 特别是共价分子间交联 胶原蛋白分子之间的相互作用被认为是 的稳定性，生物力学和理化特性， 胶原纤维基质。 最近有很多关于 I型胶原交联的位置和化学性质，但非常 人们对其功能知之甚少。 我们建议将 特定I型胶原交联的功能作用，通过改变 前α 2（I）中特定分子位点的氨基酸残基 胶原蛋白链，并评估对I型胶原蛋白的影响 在体内和体外各种组织中的结构/功能。 这么做 将产生高效的前α 2（I）胶原蛋白表达基因 和氨基酸取代：赖氨酸-5N（氨基端肽：主要 骨架交联）、组氨酸-92（螺旋：皮肤特异性交联），以及 羟辛-87（螺旋：骨骼和软组织交联）。 这些 将构建体显微注射到受精的OIM（对于空的纯合的 COLIA 2等位基因）小鼠卵母细胞产生转基因小鼠，oim-a2（I） [转基因野生型]和交联负[oim-alpha 2（I）-lys-5 N，oim- α 2（I）-his-92和oim-α 2（I）-hyl-87）]。 成效为何 转基因在体内的表达将通过几个参数来表征： 形态学，组织特异性表达，交联形成，成骨率 皮肤、骨和肌腱的形成和生物力学强度。 到 体外评价交联的组织特异性生化作用 原代成骨细胞（矿化）和皮肤成纤维细胞（非矿化） 将建立来自这些转基因OIM小鼠的培养物， 能够积聚基质、交联和矿化胶原 矩阵将被确定。 理解特定交联的作用 在不同的组织中是理解结构/功能的关键 胶原蛋白在纤维结构中可能起着重要作用， 我们对某些胶原蛋白紊乱的发病机制的理解， 肌肉骨骼系统的老化。

Modifications of specific amino acids in the pro alpha chains of type I collagen are thought to be major contributor to the biochemical, physical, and biomechanical properties of the type I collagen fibrillar architecture, dictating specialized functions in mineralized and nonmineralized tissues. In particular covalent intermolecular crosslinks between collagen molecules are postulated to be principal determinants of the stability, biomechanical and physiochemical properties of the collagen fibrillar matrix. Much has recently been elucidate concerning the location and chemical nature of type I collagen crosslinks, but very little is understood about their function. We propose to define the functional role of specific type I collagen crosslinks by altering the amino acid residues at specific molecular loci in the proalpha2(I) collagen chain and evaluating the effects on type I collagen structure/function in vivo and in vitro in various tissues. To do this high efficiency proalpha2(I) collagen expression genes will be created and amino acid substitutions: lysine-5N (amino-telopeptide: major skeletal crosslink), histidine-92 (helix: skin-specific crosslink), and hydroxylsine-87 (helix: skeletal and soft tissue crosslink). These constructs will be microinjected into fertilized oim (homozygous for null COLIA2 allele) mouse oocytes to generate transgenic mice, oim-a2(I) [transgenic wildtype] and cross-link minus [oim-alpha2(I)-lys-5N, oim- alpha2(I)-his-92, and oim-alpha2(I)-hyl-87)]. The effects of these transgenes in vivo will be characterized by several parameters: gross morphology, tissue specific expression, crosslink formation, rate of bone formation, and biomechanical strength of skin, bone and tendon. To evaluate in vitro the tissue specific biochemical role of crosslinks primary osteoblast (mineralizing) and skin fibroblast (non-mineralizing) cultures from these transgenic oim mice will be established and their ability to accumulate matrix, crosslink, and mineralize collagenous matrix will be determined. Understanding the role of specific crosslinks in different tissues is essential to understanding the structure/function of collagen in the fibrillar architecture may play a significant role in our understanding the pathogenesis of certain collagen disorders, and aging of the musculoskeletal system.