CARDIOVASCULAR DEVELOPMENT IN XENOPUS LAEVIS

非洲爪蟾的心血管发育

• 批准号: 6413002
• 负责人: DANIEL L. WEEKS
• 金额: $ 44.31万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6413002
• 关键词: DNA binding protein; Xenopus; Xenopus oocyte; antisense nucleic acid; cardiovascular system; developmental genetics; early embryonic stage; fluorescence microscopy; gene expression; genetically modified animals; green fluorescent proteins; histogenesis; linkage mapping; nonmammalian vertebrate embryology; transfection /expression vector; transforming growth factors; vascular endothelial growth factors; vascular endothelium

(Adapted from the Applicant's Abstract) The rapid progress in studies that identify the genetic basis for congenital defects has increased the urgency for rapid and cost effective ways to manipulate specific gene expression during vertebrate development. One of the best studied vertebrates, Xenopus laevis offers many advantages for the examination of early cardiac development. Methods to make transgenic frog embryos provide an opportunity to look specifically at the control of cardiovascular gene expression using transgenic vectors that report the control of transcription by expression of green fluorescent protein (GFP). These vectors will be constructed to use either mammalian and amphibian promoters to examine conservation of control of tissue and temporal specific gene expression. GFP expression transgenic embryos can be monitored by fluorescence microscopy without sacrificing the transgenic embryo. Three specific genes, already implicated in cardiovascular defects or development will also be examined using the well established method of mRNA and antisense oligonucleotide injection into Xenopus embryos. Msx-1, a homeobox containing repressor protein, is implicated in Down's Syndrome related atrial-ventricular septal defects (AVSD). The hypothesis that the important balance of Msx-1 levels with the genes it regulates is disrupted in some Down's individuals will be tested by deliberate modification of the levels of the Msx-1 homologue in Xenopus embryos. Betaglycan, the TGFbeta-2 binding protein is found in a small region of chromosome 1 associated with some cases of non-Down's syndrome related AVSD. The investigators will test the hypothesis that specific mutations or altered levels of betaglycan in the heart lead to valvular defects. Finally, the investigators will investigate the control of vascular endothelial growth factor (VEGF). VEGF is an essential signaling molecule for the establishment and maintenance of vascular tissue. The presence of VEGF mRNA in frog oocytes, coupled with the ease of oocyte manipulation provides an opportunity to examine VEGF control in single cell assays. The free living, very visible development of the Xenopus embryo will provide a means to examine the roles of different VEGF isoforms and the activation of VEGF during cardiovascular development. The investigators will also examine the effect of hypoxia on VEGF expression to begin to develop a way to assay how environmental conditions may lead to defects ev en when no specific mutation is present.

（改编自申请人的摘要）鉴定先天性缺陷的遗传基础的研究的快速进展增加了在脊椎动物发育期间操纵特异性基因表达的快速且成本有效的方法的紧迫性。 非洲爪蟾是研究得最好的脊椎动物之一，它为研究早期心脏发育提供了许多优势。 制备转基因青蛙胚胎的方法提供了一个机会，利用转基因载体来具体观察心血管基因表达的控制，所述转基因载体报告通过表达绿色荧光蛋白（GFP）来控制转录。将构建这些载体以使用哺乳动物和两栖动物启动子来检查组织和时间特异性基因表达的控制的保守性。 GFP表达转基因胚胎可以通过荧光显微镜监测而不牺牲转基因胚胎。三个特定的基因，已经牵连在心血管缺陷或发展也将使用mRNA和反义寡核苷酸注射到非洲爪蟾胚胎的成熟的方法进行检查。Msx-1是一种含有抑制蛋白的同源盒，与唐氏综合症相关的房室间隔缺损（AVSD）有关。 Msx-1水平与其调控的基因的重要平衡在某些唐氏个体中被破坏的假设将通过故意修改非洲爪蟾胚胎中Msx-1同源物的水平来进行测试。 β聚糖，TGF β-2结合蛋白，发现于与一些非唐氏综合征相关的AVSD病例相关的1号染色体的一个小区域。 研究人员将检验心脏中β聚糖的特定突变或水平改变导致瓣膜缺陷的假设。 最后，研究者将研究血管内皮生长因子（VEGF）的控制。VEGF是血管组织建立和维持所必需的信号分子。 青蛙卵母细胞中VEGF mRNA的存在，加上卵母细胞操作的容易性，提供了一个机会，检查VEGF控制在单细胞测定。 非洲爪蟾胚胎的自由生活，非常明显的发展将提供一种手段，以检查不同的VEGF亚型的作用和血管内皮生长因子在心血管发育过程中的激活。 研究人员还将检测缺氧对VEGF表达的影响，开始开发一种方法来分析环境条件如何导致缺陷，即使不存在特定突变。