MOLECULAR AND GENETIC BASIS OF CONOTRUNCAL MORPHOGENESIS

该项目在《SCOR》中有两个主要目标。第一个是 确定项目1中确定的人类基因的相关性和 分离于项目2至圆锥体干形态发生。这将是 通过首先确定表达的程序和位置来完成 小鼠胚胎发育过程中候选基因或遗传物质的RT- 聚合酶链式反应扩增、原位杂交和免疫化学。这个 任何候选基因对圆锥干发育的功能意义 将通过使用反义阻断基因表达来确定 脱氧核糖核苷酸外用、局部注射或 在培养的小鼠胚胎中使用病毒载体产生细胞内 病毒感染后不适当的基因表达 携带全长cDNAs的载体。圆锥主干或 血管的形成将被解剖和监测 心内膜特异性单抗的免疫组织化学研究 和内皮细胞。第二个主要目标是独立识别和 如上所述，表征圆锥主干过程中表达的基因 心、颅神经脊及内的形态发生 对照组和对照组的咽弓和心脏流出道 突变的小鼠胚胎。这将通过差分来实现 RT-PCR扩增产物在测序凝胶上的比较 通过聚合酶链式反应扩增、RACE扩增和小鼠基因文库筛选 库来产生用于功能分析的探针。的行为 来自突变小鼠胚胎的脑神经和心脏神经脊将是 通过将细胞移植到突变或对照培养的小鼠中进行评估 胚胎和小鸡胚胎一样。这项工作完成后，我们将 已经确定了任何候选人类基因的功能意义 到圆锥干形态发生以及独立鉴定的新的 在小鼠心脏发育早期表达的基因可以是 用作人类材料的细胞遗传学和分子研究的探针 从而开始解开基因表达和功能的程序 为正确的编队和集结而系统地发挥 心脏的圆锥干区。

This project has two major goals within the SCOR. The first is to determine the relevance of human genes identified in Project 1 and isolated in Project 2 to conotruncal morphogenesis. This will be accomplished by first determining the program and site of expression of candidate genes or genetic material in the developing mouse embryo by RT- PCR amplification, in situ hybridization and immunochemistry. The functional significance of any candidate gene to conotruncal development will be determined by blocking gene expression using anti-sense deoxyribonucleotides administered externally, injected locally or generated intracellular using viral vectors in cultured mouse embryos and by inappropriate gene expression following administration of viral vectors carrying full-length cDNA's. Disruption of conotruncal or vascular formation will be monitored anatomically and immunohistochemically using monoclonal antibodies specific to endocardium and endothelium. The second major goal is to independently identify and characterize, as described above, gene expressed during conotruncal morphogenesis by cardiac and cranial neural crest as well as within pharyngeal arches and the outflow tract of the heart in the control and mutant mouse embryos. This will be accomplished by differential comparison of RT-PCR amplified mRNA products on sequencing gels followed by PCR amplification, RACE extension and cDNA library screening of mouse libraries to produce probes for functional analysis. The behavior of cranial and cardiac neural crest from mutant mouse embryos will be evaluated by transplanting cells into mutant or control cultured mouse embryos as well as chick embryos. Upon completion of this work we will have determined the functional significance of any candidate human genes to conotruncal morphogenesis as well as independently identified new genes expressed during early heart development in the mouse that can be used as probes for cytogenetic and molecular studies in human material thereby beginning to unravel the program of gene expression and function being systematically played out for the correct formation and assembly of the conotruncal region of the heart.