GENETIC ANALYSIS OF TYPE I COLLAGEN FUNCTION

I型原-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、生物力学特性 建筑，规定了矿化和矿化的特殊功能 非矿化组织。尤其是共价分子间交联物 胶原蛋白分子之间被认为是主要的决定因素 的稳定性、生物力学和物理化学特性 胶原纤维基质。最近关于以下问题有很多澄清 I型胶原交联物的位置和化学性质，但非常 人们对它们的功能知之甚少。我们建议定义 特异I型胶原交联物的功能作用 前α2特定分子座位上的氨基酸残基(I) 胶原链及其对I型胶原影响的评价 体内和体外各种组织的结构/功能。要做到这一点 将创造高效的前α2(I)胶原蛋白表达基因 氨基酸取代：赖氨酸-5N(氨基端肽：主要 骨骼交联物)、组氨酸-92(螺旋：皮肤特异性交联物)和 羟基氨酸-87(螺旋：骨骼和软组织交联物)。这些 构建物将被显微注射到受精的OIM中(纯合子为空 COLIA2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]和交联体减去[OIM-α2(I)-Lys-5N，OIM- Alpha2(I)-His-92和OIM-Alpha2(I)-HYL-87)]。这些因素的影响 体内转基因将由几个参数来表征：葛罗斯 形态、组织特异性表达、交联物形成、骨速率 皮肤、骨骼和肌腱的形成和生物力学强度。至 体外评价交联物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) 这些转基因OIM小鼠的培养将被建立，他们的 积累基质、交联和矿化胶原蛋白的能力 矩阵将被确定。了解特定交联剂的作用 对于理解其结构/功能是必不可少的。 纤维状结构中的胶原蛋白可能在 我们对某些胶原蛋白紊乱的发病机制的理解，以及 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.