RETROVIRAL TRANSDUCTION AND IMMORTALIZATION OF FILARIA

描述：尽管最近化疗取得了进展，但人类丝虫病 感染仍然是重要的公共卫生问题。最近，伟大的 我们对基因谱系的理解已经取得了进展 在这些寄生虫身上表达。然而，缺乏遗传方法 操纵和寄生虫材料的匮乏已经并将继续下去 限制对这些生物的研究。最近，有证据表明， 外源DNA序列可被导入丝虫的胚胎 马来丝虫(Brugia Malayi)的生物学方法及泛嗜性转基因 逆转录病毒载体。这项提案的总体目标将是利用这些 克服缺乏传统遗传学所带来的限制的系统 以及无法获得大量寄生虫材料。具体的 这项建议的目的是： 1.应用生物瞬时转染系统鉴定作用中的顺式细胞 影响马来不动杆菌基因表达的重要因素。参数应为 探索将包括识别瞬变的最小启动子 5‘和3’序列在马来不动杆菌中的表达 信息处理，以及合成内含子的添加的影响。 2.建立泛嗜性逆转录病毒载体作为稳定外源基因的系统 在马来丝虫胚胎中的表达。 3.利用表达癌基因的泛嗜性逆转录病毒载体创建 马来丝虫永生细胞系。 4.确定转基因马来丝虫胚胎中是否存在异源DNA 稳定遗传的。

DESCRIPTION: Despite recent advances in chemotherapy, the human filarial infections remain important public health problems. Recently, the great advances have been made in our understanding of the repertoire of genes expressed in these parasites. However, the lack of methods of genetic manipulation and the paucity of parasite material has limited and will continue to limit studies of these organisms. Recently, it has been demonstrated that exogenous DNA sequences can be introduced into embryos of the filarial parasite Brugia malayi, employing biolistic methods and transfection with pantropic retroviral vectors. The overall goal of this proposal will be to employ these systems to overcome the limitations imposed by the lack of traditional genetics and the inability to obtain large amounts of parasite material. The specific aims of this proposal are: 1. To employ the biolistic transient transfection system to identify cis acting factors important in controlling gene expression in B. malayi. Parameters to be explored will include identification of a minimal promoter for transient expression in B. malayi, the effect of 5' and 3' sequences important for message processing, and the effect of the addition of synthetic introns. 2. To develop pantropic retroviral vectors as a system for stable foreign gene expression in B. malayi embryos. 3. To employ pantropic retroviral vectors expressing oncogenes to create an immortalized cell line from B. malayi. 4. To determine if heterologous DNA present in transfected B. malayi embryos is stably inherited.