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）研究中确定先天性缺陷的遗传基础的快速进步提高了在脊椎动物发育过程中操纵特定基因表达的快速和成本效益方法的紧迫性。 Xenopus laevis是最精细的脊椎动物之一，为检查早期心脏发展提供了许多优势。 使转基因青蛙胚胎的方法提供了一个机会，可以使用转基因载体专门研究心血管基因表达的控制，这些向量通过表达绿色荧光蛋白（GFP）来报告转录控制。这些载体将被构造为使用哺乳动物和两栖动物启动子检查组织控制和时间特异性基因表达的保护。 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。还将使用已建立的mRNA和反义寡核苷酸注射到Xenopus胚胎中的三个特定基因，与心血管缺陷或发育有关。 MSX-1是一种含有抑制剂蛋白的同源蛋白质，与唐氏综合症相关的心房间隔缺陷（AVSD）有关。 通过故意修改Xenopus胚胎中MSX-1同源物的水平，将对某些人的个体中的MSX-1水平与它所调节的基因的重要平衡与所调节的基因的重要平衡进行。 betaglycan，TGFBETA-2结合蛋白在与某些与某些非下降综合征相关的AVSD相关的染色体的小区域中发现。 研究者将检验以下假设：心脏中特定突变或β地糖的水平改变导致瓣膜缺陷。 最后，研究人员将研究血管内皮生长因子（VEGF）的控制。 VEGF是用于建立和维持血管组织的必不可少的信号分子。 青蛙卵母细胞中VEGF mRNA的存在，再加上卵母细胞操纵的易感性，为检查单细胞测定中的VEGF控制提供了机会。 Xenopus胚胎的自由生活，非常明显的发展将提供一种检查不同VEGF同工型的作用以及在心血管发育过程中VEGF的激活的方法。 研究者还将检查缺氧对VEGF表达的影响，以开始开发一种方法来分析环境条件可能导致缺陷EV EV EV EN时，当不存在特定的突变时。