Targeted Disruption--Mouse VEGF AURE Regulatory Domain

描述（由申请人提供）：在氧张力降低的条件下 组织血管内皮生长因子（VEGF）的产生 增加，导致新生血管形成。i）关节炎关节， 类风湿性关节炎，ii）动脉粥样硬化斑块，或iii）肿瘤。在我们的模型中 在转录后调节中，VEGF的表达受到以下两种因素的控制： mRNA稳定性增加或从翻译抑制中释放。的 在我们的模型中，介导VEGF产生增加的因素是：i） 作用于，ii）顺式富含AU的反应的反式作用RNA结合蛋白 VEGF mRNA的VEGF中的AURE元件。我们建议描述的作用， f通过进行AURE的靶向破坏，在VEGF 31 UTR中的AURE 在小鼠的VEGF基因座中的调节结构域。使用这种方法，我们将 产生培养的细胞系和小鼠，其中VEGF的产生是 与转录后调节机制分离。 本提案中的具体目标将评估VEGF的贡献 AURE结构域如下：在Aim I中，我们将产生靶向载体， 同源重组，并使用它来破坏VEGF中的AURE结构域， 小鼠胚胎干细胞（ES细胞）中的VEGF。这种干扰不会影响 VEGF基因座的编码区，但由于AURE往往是不稳定的，我们 推测VEGF表达可能上调。在目标2中，我们将确定 VEGF AURE的靶向破坏对以下产生表型效应：i） VEGF产生，或ii）ES细胞在裸鼠中的致瘤性。在目标3 我们将询问在VEGF AURE突变体中测量的表型变化是否由以下因素引起： VEGF mRNA的周转或翻译抑制的变化。最后在 目的4，我们将使用VEGF AURE突变体细胞来创建纯合和 杂合子小鼠品系，并确定VEGF过度产生对 胚胎发生和成年小鼠的生理学和病理生理学。述VEGF AURE突变体细胞系的发展，在这项建议将是非常宝贵的研究 胚胎发生，如果产生VEGF AURE突变小鼠，这些小鼠将 提供了一个令人兴奋的系统，研究血管过度，因为发现 在关节炎关节，肿瘤和增生性视网膜病变，并研究 VEGF对免疫细胞的影响，包括单核细胞和破骨细胞， 关节炎

DESCRIPTION (provided by applicant): Under conditions of reduced oxygen tension (hypoxia) production of tissue vascular endothelial growth factor (VEGF) increases, resulting in neovascularization of. i) arthritic joints in rheumatoid arthritis, ii) atherosclerotic plaques, or iii) tumors. In our model of post-transcriptional regulation, VEGF expression is controlled either by increases in mRNA stability or by release from translational repression. The factors, in our model, that mediate increased VEGF production are: i) trans-acting RNA binding proteins that act on, ii) cis AU-rich response elements (AURE) in the VEGF of VEGF mRNA. We propose to characterize the role o f AURE in the VEGF 31UTR by performing targeted disruption of the AURE regulatory domain in the VEGF locus of the mouse. Using this approach we will produce both cultured cell lines and mice in which production of VEGF is segregated from mechanisms of post-transcriptional regulation. The Specific Aims in this proposal will evaluate the contribution of the VEGF AURE domain as follows: In Aim I we will produce a targeting vector for homologous recombination and use it to disrupt the AURE domain in the VEGF of VEGF in mouse embryonic stem (ES) cells. This disruption will not affect the coding region of the VEGF locus, but because AURE are often de-stabilizing, we suspect that VEGF expression may be up-regulated. In Aim 2 we will determine if the targeted disruption of the VEGF AURE produces phenotypic effects on: i) VEGF production, or ii) the tumorigenicity of ES cells in nude mice. In Aim 3 we will ask if the phenotypic changes measured in VEGF AURE mutants result from changes in the turnover or translational repression of VEGF mRNA. Finally, in Aim 4, we will use the VEGF AURE mutant cells to create homozygous and heterozygous mouse strains and determine the effects of VEGF overproduction on embryogenesis and on the physiology and pathophysiology of adult mice. The VEGF AURE mutant cell lines developed in this proposal will be invaluable in studies of embryogenesis and, if VEGF AURE mutant mice are produced, these mice will provide an exciting system with which to study overvascularization, as is found in arthritic joints, tumors and proliferative retinopathy, and to study the effects of VEGF on immune cells, including monocytes and osteoclasts in arthritic disease.