SCREEN FOR LYMPHOID MUTANTS

筛查淋巴细胞突变体

• 批准号: 6952138
• 负责人: NIKOLAUS S TREDE
• 金额: $ 12.43万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6952138
• 关键词: artificial chromosomes; chromosome walking; developmental genetics; developmental immunology; gene mutation; genetic mapping; genetic screening; histogenesis; in situ hybridization; lymphopoiesis; nonmammalian vertebrate embryology; pharynxs; regulatory gene; thymus; zebrafish

During lymphopoiesis a pluripotent stem cell develops into T or B lymphocytes. The thymus provides a specialized environment for terminal T lymphocyte differentiation. By studying natural mouse mutants and "knock-out" mice, several of the important genes in lymphocyte development and thymic organogenesis have been recently characterized. A full understanding of lymphopoiesis would also require the identification and hierarchical ordering of very early regulatory genes. To achieve this understanding we propose to take a novel approach using the zebrafish (Danio rerio) as a genetic model system. The zebrafish combines genetic and embryologic advantages and thus offers an ideal model system to study lymphopoiesis. A mutagenesis screen was done using chemical mutagenesis and ionizing radiation. F2 progeny from mutagenized fish were screened with probes for the recombination activating gene-1 (Rag-1) and alpha embryonic globin by whole mount in-situ hybridization. Of 17 potential mutants with absent Rag-1 staining in the thymus, eight were confirmed to date by F2 incrossing. One of these mutants, CZ-3, is an autosomal recessive mutation which results in microcephaly, microphthalmia and abnormalities of the pharyngeal arches. Lymphoid markers, including the earliest gene in lymphoid ontogeny Ikaros, are totally absent in thymi from CZ-3. Histologic sections show a rudimentary thymus and complete absence of lymphocytes by ultrastructural analysis. Other hematopoietic lineages are normal in CZ-3. I have undertaken a positional cloning strategy to identify the CZ-3 gene. I have mapped the mutated gene with a closely linked marker, z10517 (0.33 cM based on 9/2744 meiotic recombinants) and have established a YAC contig distal to z10517. The zebrafish homolog of whn, the nude mouse gene, colocalizes with z10517 on the same YAC clone. whn is an attractive candidate for the gene mutated in CZ-3, as the immune deficiency in the nude mouse is the result of a rudimentary thymus. A specific aim of this proposal is to test if CZ-3 and whn are identical. If they are not, I will continue the chromosomal walk and clone CZ-3 by genetic and functional means. If CZ-3 and whn are identical I will map the remaining mutants. I will select two mutants with interesting phenotypes (which fall into two groups: those with normal and with abnormal pharyngeal arches) for fine-mapping. Cloning of these genes will further our understanding of phylogeny of the immune system as well as of molecular events in both normal T lymphopoiesis and diseases such as immune deficiencies and leukemias and will be instrumental in devising novel therapeutic strategies.

在淋巴细胞生成过程中，多能干细胞发育成 T 或 B 淋巴细胞。 胸腺为终末T淋巴细胞分化提供了特殊的环境。 通过研究天然小鼠突变体和“基因敲除”小鼠，最近对淋巴细胞发育和胸腺器官发生中的几个重要基因进行了表征。 对淋巴细胞生成的充分理解还需要对非常早期的调控基因进行识别和分层排序。 为了实现这一理解，我们建议采用一种新方法，使用斑马鱼（Danio rerio）作为遗传模型系统。 斑马鱼结合了遗传和胚胎学优势，从而为研究淋巴细胞生成提供了理想的模型系统。 使用化学诱变和电离辐射进行诱变筛选。 通过整体原位杂交，用重组激活基因 1 (Rag-1) 和 α 胚胎球蛋白探针筛选诱变鱼的 F2 后代。 胸腺中缺乏 Rag-1 染色的 17 个潜在突变体中，迄今为止通过 F2 近交证实了 8 个突变体。 其中一种突变体 CZ-3 是一种常染色体隐性突变，可导致小头畸形、小眼畸形和咽弓异常。 淋巴标记，包括淋巴个体发育中最早的基因 Ikaros，在 CZ-3 的胸腺中完全不存在。 通过超微结构分析，组织学切片显示未发育的胸腺和完全缺乏淋巴细胞。 CZ-3 中其他造血谱系正常。 我采取了定位克隆策略来鉴定 CZ-3 基因。 我用紧密连锁的标记 z10517（0.33 cM，基于 9/2744 减数分裂重组体）绘制了突变基因的图谱，并建立了 z10517 远端的 YAC 重叠群。裸鼠基因whn的斑马鱼同源物与z10517共定位于同一YAC克隆上。 whn 是 CZ-3 突变基因的一个有吸引力的候选者，因为裸鼠的免疫缺陷是胸腺发育不全的结果。 该提案的一个具体目的是测试 CZ-3 和 whn 是否相同。 如果不是，我将继续染色体行走并通过遗传和功能手段克隆 CZ-3。 如果 CZ-3 和 whn 相同，我将绘制其余突变体的图谱。 我将选择两个具有有趣表型的突变体（分为两组：具有正常咽弓的突变体和具有异常咽弓的突变体）进行精细绘图。 这些基因的克隆将进一步了解免疫系统的系统发育以及正常 T 淋巴细胞生成和免疫缺陷和白血病等疾病中的分子事件，并将有助于设计新的治疗策略。

项目成果

A model 450 million years in the making: zebrafish and vertebrate immunity.

• DOI: 10.1242/dmm.007138
• 发表时间: 2012-01
• 期刊: Disease models & mechanisms
• 影响因子: 4.3
• 作者: Renshaw SA;Trede NS
• 通讯作者: Trede NS

Trans-centromere effects on meiotic recombination in the zebrafish.

跨着丝粒对斑马鱼减数分裂重组的影响。

• DOI: 10.1534/genetics.110.124081
• 发表时间: 2011
• 期刊: Genetics
• 影响因子: 3.3
• 作者: Demarest,BradleyL;Horsley,WyattH;Locke,ErinE;Boucher,Kenneth;Grunwald,DavidJ;Trede,NikolausS
• 通讯作者: Trede,NikolausS