CARDIOVASCULAR DEVELOPMENT IN XENOPUS LAEVIS

非洲爪蟾的心血管发育

• 批准号: 6565114
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565114
• 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)确定先天性缺陷的遗传基础的研究的快速进展增加了对在脊椎动物发育过程中操纵特定基因表达的快速且成本有效的方法的紧迫性。 非洲爪蟾是研究最深入的脊椎动物之一，为检查早期心脏发育提供了许多优势。 制作转基因青蛙胚胎的方法提供了一个机会，可以利用转基因载体来专门研究心血管基因表达的控制，这些转基因载体通过绿色荧光蛋白（GFP）的表达来报告转录的控制。这些载体将被构建为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控制的保守性。 可以通过荧光显微镜监测GFP表达转基因胚胎而不牺牲转基因胚胎。还将使用将 mRNA 和反义寡核苷酸注射到非洲爪蟾胚胎中的成熟方法来检查已经与心血管缺陷或发育有关的三个特定基因。 Msx-1 是一种含有阻遏蛋白的同源盒，与唐氏综合症相关的房室间隔缺损 (AVSD) 有关。 一些唐氏个体中 Msx-1 水平与其调节基因的重要平衡被破坏的假设将通过对非洲爪蟾胚胎中 Msx-1 同源物水平的有意修改进行检验。 Betaglycan（TGFbeta-2 结合蛋白）存在于 1 号染色体的一个小区域，与一些非唐氏综合症相关的 AVSD 病例相关。 研究人员将验证心脏中β聚糖的特定突变或水平改变会导致瓣膜缺陷的假设。 最后，研究人员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的重要信号分子。 青蛙卵母细胞中存在 VEGF mRNA，加上卵母细胞操作的简便性，为在单细胞测定中检查 VEGF 控制提供了机会。 非洲爪蟾胚胎的自由生活、非常明显的发育将为检查不同 VEGF 亚型的作用以及 VEGF 在心血管发育过程中的激活提供一种方法。 研究人员还将检查缺氧对 VEGF 表达的影响，以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。