Mechanisms Of Lineage-specific Gene Expression

谱系特异性基因表达机制

• 批准号: 7194124
• 负责人: JOHN H KEHRL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7194124
• 关键词: B lymphocyte; apoptosis; cytogenetics; developmental genetics; developmental immunology; developmental neurobiology; gene expression; gene targeting; genetic transcription; genetically modified animals; histogenesis; human tissue; immunocytochemistry; laboratory mouse; microarray technology; motor neurons; northern blottings; nucleic acid probes; pancreas; polymerase chain reaction; site directed mutagenesis; southern blotting; transcription factor

This project focuses on the transcription factors HB9 and BSAP, originally identified in B lymphocytes. The HB9 gene also referred to as HlxB9 encodes a homeodomain coding protein. Gene targeting in mice has revealed a critical role for HB9 in motor neuron and pancreas development. Abnormalities in the sacral region occurs in humans with one abnormal HB9 allele. Mice heterozygotic for an HB9 null mutation are normal, but mice that lack HB9 die at birth of respiratory failure due to a lack of diaphragm innervation. The motor neuron defects may arise from the inappropriate expression of genes normally expressed in other types of neurons. These mice also lack a portion of their pancreas, the dorsal lobe, and have reduced numbers of insulin producing beta cells in their residual ventral lobe. Because the developmental defects and perinatal lethality makes assesmment of the function of HB9 in adult tissues impossible, we have used a conditional gene targeting approach and have introduced loxP sites on either side of exon 3 of the HB9 gene. We have shown that the introduction of the LoxP sites does not interfere with normal HB9 expression. We have obtained MX-Cre transgenic mice, where the Cre recombinase can be induced by interferon treatment. We have introduced the MX-Cre transgene onto the HB9 loxP background. We have shown that the expression of Cre causes the deletion of exon 3 of HB9, but good deletion only occured in spleen, bone marrow, and intestine. In contrast, we found little deletion in the pancreas. We have found little effect on deleting HB9 in those tissues where Cre is expressed. To determine the consequences of deleting HB9 in the developing and adult pancrease we have crossed the HB9 Cre lox mice with mice expressing Cre from a transgene regulated by the insulin promoter. Mice in which HB9 exon 3 is flanked by loxP sites on both alleles and which express Cre from the insulin promoter should be available this fall for analysis. BSAP is a transcription factor critical for B-lymphocyte development and lineage committment. Mice overexpressing BSAP have B cells which are hyperproliferative, have an apoptosis defect, and a propensity for developing lymphomas. We have largely completed the analysis of these mice.

该项目重点关注最初在 B 淋巴细胞中发现的转录因子 HB9 和 BSAP。 HB9基因也称为HlxB9，编码同源域编码蛋白。小鼠基因靶向研究揭示了 HB9 在运动神经元和胰腺发育中的关键作用。具有一种异常 HB9 等位基因的人类会出现骶骨区域的异常。 HB9 无效突变的杂合小鼠是正常的，但缺乏 HB9 的小鼠在出生时会因缺乏膈肌神经支配而死于呼吸衰竭。运动神经元缺陷可能是由于通常在其他类型神经元中表达的基因的不适当表达引起的。这些小鼠还缺乏一部分胰腺，即背叶，并且残余腹叶​​中产生胰岛素的β细胞数量减少。由于发育缺陷和围产期致死率使得无法评估 HB9 在成人组织中的功能，因此我们使用了条件基因靶向方法，并在 HB9 基因外显子 3 的两侧引入了 loxP 位点。我们已经证明 LoxP 位点的引入不会干扰正常的 HB9 表达。我们已经获得了MX-Cre转基因小鼠，其中Cre重组酶可以通过干扰素治疗来诱导。我们已将 MX-Cre 转基因引入到 HB9 loxP 背景上。我们已经证明Cre的表达导致HB9外显子3的缺失，但良好的缺失仅发生在脾、骨髓和肠中。相比之下，我们发现胰腺几乎没有缺失。我们发现在表达 Cre 的组织中删除 HB9 几乎没有影响。为了确定在发育中和成年胰腺中删除 HB9 的后果，我们将 HB9 Cre lox 小鼠与表达由胰岛素启动子调节的转基因的 Cre 的小鼠杂交。 HB9 外显子 3 的两个等位基因两侧均带有 loxP 位点且表达胰岛素启动子 Cre 的小鼠将于今年秋季用于分析。 BSAP 是一种对于 B 淋巴细胞发育和谱系定型至关重要的转录因子。过度表达 BSAP 的小鼠的 B 细胞过度增殖，具有细胞凋亡缺陷，并且有发展为淋巴瘤的倾向。我们已经基本完成了对这些小鼠的分析。