SCREEN FOR LYMPHOID MUTANTS

筛查淋巴细胞突变体

• 批准号: 6499114
• 负责人: NIKOLAUS S TREDE
• 金额: $ 12.46万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 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 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6499114
• 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淋巴细胞的分化提供了一个特殊的环境。 通过对自然突变小鼠和基因敲除小鼠的研究，淋巴细胞发育和胸腺器官发生中的几个重要基因最近已被鉴定。 对淋巴细胞生成的全面了解还需要对非常早期的调控基因进行鉴定和分级排序。 为了实现这种理解，我们建议采取一种新的方法，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，因此提供了研究淋巴细胞生成的理想模型系统。 使用化学诱变和电离辐射进行诱变筛选。 用重组激活基因-1（Rag-1）和α-胚胎珠蛋白的探针通过整装原位杂交筛选来自诱变鱼的F2后代。 在胸腺中缺少Rag-1染色的17个潜在突变体中，迄今为止通过F2交叉确认了8个。 其中一种突变体CZ-3是常染色体隐性突变，可导致小头畸形、小眼畸形和咽弓异常。 类胸腺标记，包括淋巴个体发育中最早的基因Ikaros，在CZ-3胸腺中完全不存在。 组织切片显示胸腺发育不全，超微结构分析显示淋巴细胞完全缺失。 其他造血谱系在CZ-3中是正常的。 我已经采取了定位克隆策略来识别CZ-3基因。 我已经绘制了突变基因与紧密连锁的标记，z10517（0.33 cM的基础上9/2744减数分裂重组），并建立了一个YAC重叠群远端z10517。斑马鱼同源whn，裸鼠基因，共定位与z10517上相同的YAC克隆。 whn是CZ-3中突变基因的有吸引力的候选者，因为裸鼠中的免疫缺陷是胸腺发育不全的结果。 该提案的一个具体目标是测试CZ-3和whn是否相同。 如果没有，我将继续染色体步移，通过遗传和功能手段克隆CZ-3。 如果CZ-3和whn是一样的我就能绘制出剩下的变异体。 我将选择两个突变体有趣的表型（分为两组：正常和异常咽弓）进行精细定位。 这些基因的克隆将进一步加深我们对免疫系统的遗传学以及正常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的理解，并将有助于设计新的治疗策略。