MODELING DIAMOND-BLACKFAN ANEMIA

塑造钻石-黑粉丝贫血症

• 批准号: 6954695
• 负责人: Thomas M. Glaser
• 金额: $ 14.77万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6954695
• 关键词: alleles; aminohydrolases; biomarker; blood cell count; cell differentiation; cell line; cell proliferation; congenital aplastic anemia; disease /disorder etiology; disease /disorder model; embryonic stem cell; erythropoietin; flow cytometry; gene expression; gene mutation; genetic models; genetically modified animals; hematopoiesis; hemoglobin F; laboratory mouse; model design /development; phenylhydrazines; reticulocytes; ribosomal proteins; transposon /insertion element

DESCRIPTION (provided by applicant): One-quarter of families with Diamond-Blackfan anemia (DBA) have heterozygous mutations in RPS19, which encodes the 40S ribosomal subunit protein S19 (Willig et at. 1999). Children with DBA have a nonregenerative red blood cell aplasia with variable birth defects, including growth retardation, craniofacial malformations, triphalangeal thumbs and preaxial polydactyly. In view of the universal cellular requirement for ribosomes, the tissue specificity of RPS19 phenotypes is puzzling. The mechanism of erythroid marrow failure in DBA is unknown. In Drosophila, riboprotein mutations, termed Minutes, are abundant and dispersed across the genome (Lambertsson 1998). They are homozygous lethal and have similar heterozygous phenotypes consisting of smaller, thinner bristles and profoundly delayed development (Schultz 1929). Minutes were instrumental in early studies that defined concepts of cell autonomy, clonal lineage and developmental compartmentation, as Minute cells compete poorly with wild type clones in somatic mosaics. Apart from human DBA, little is known about ribosomal protein (RP) gene mutations in vertebrates. Recently, my laboratory discovered that the spontaneous mouse mutation Bst (belly spot and tail) is caused by an intragenic deletion in Rpl24, which encodes the 60S ribosomal subunit protein L24 (Oliver et al. 2004). Bst/+ mice have vertebral defects (tail kinks), preaxial polydactyly, midventral spotting and a deficiency of retinal ganglion cells. Homozygotes die before implantation. The Bst mutation causes aberrant splicing in 80% of Rpl24 transcripts, truncation of L24 protein, and delayed ribosome biogenesis in vivo. Bst/+ fibroblasts grow slower than wild type, remain longer in G1 phase, and exhibit decreased overall rates of protein synthesis. Mouse BAG and human cDNA transgenes correct this phenotype. The hematopoietic capacity of Bst/+ mice has not been explored in detail. In this proposal, we aim to: (1) generate mice with null and hypomorphic mutations in Rps19, using selected gene trap ES cell lines; (2) characterize hematopoiesis in Rps19 knockout mice and the spontaneous riboprotein mutants Bst/+ (Rpl24) and Ts/+ (Rpl38), under normal and erythroid stress conditions; and (3) test two alternative mechanisms for the marrow specificity of DBA mutations - that normal erythroid progenitors express relatively low levels of Rps19 mRNA, making them particularly sensitive to reduced gene dosage, and that S19 has an extraribosomal function to promote red cell differentiation.

描述（由申请人提供）： 有四分之一的钻石 - 黑色贫血（DBA）家族在RPS19中具有杂合突变，该突变编码40S核糖体亚基蛋白S19（Williget。1999）。 DBA儿童患有非再生性的红细胞性植物，具有可变的先天缺陷，包括生长迟缓，颅面畸形，三角司朗格朗的拇指和先前的多态度。鉴于核糖体的通用细胞需求，RPS19表型的组织特异性令人困惑。 DBA中红细胞骨髓衰竭的机制尚不清楚。在果蝇中，核蛋白突变（称为分钟数分钟）丰富并且在基因组中分散（Lambertsson 1998）。它们是纯合致死的，具有相似的杂合表型，包括较小，较薄的刷毛和深刻延迟的发育（Schultz 1929）。在早期研究中，分钟数分钟有助于定义细胞自主性，克隆谱系和发育隔室的概念，因为微小细胞与体细胞马赛克中的野生型克隆竞争不佳。除了人DBA外，脊椎动物中核糖体蛋白（RP）基因突变知之甚少。最近，我的实验室发现，自发小鼠突变BST（腹部斑点和尾巴）是由RPL24中的基因缺失引起的，该缺失编码了60年代的核糖体亚基蛋白L24（Oliver等，2004）。 BST/+小鼠具有椎骨缺陷（尾扭结），术前多态，室内斑点和视网膜神经节细胞缺乏。纯合子在植入前死亡。 BST突变会在80％的RPL24转录物，L24蛋白的截断和体内延迟的核糖体生物发生中引起异常剪接。 BST/+成纤维细胞的生长比野生型慢，在G1期保持更长的时间，并且蛋白质合成的总体速率降低。鼠标袋和人cDNA转基因纠正该表型。尚未详细探讨BST/+小鼠的造血能力。在此提案中，我们的目的是：（1）使用选定的基因诱使ES细胞系在RPS19中生成无效的RPS19中的小鼠； （2）在正常和红细胞应力条件下，在RPS19基因敲除小鼠和自发性核糖蛋白突变体BST/+（RPL24）和TS/+（RPL38）中表征造血症； （3）测试了DBA突变的骨髓特异性的两种替代机制 - 正常的红细胞祖细胞表达的RPS19 mRNA水平相对较低，这使得它们对降低的基因剂量特别敏感，并且S19具有促促肌体功能以促进红细胞的分化。