RETROVIRAL TRANSDUCTION AND IMMORTALIZATION OF FILARIA

描述：尽管最近在化学疗法方面取得了进展，但人类丝虫 感染仍然是重要的公共卫生问题。最近，伟大的 我们对基因库的理解已经取得了进展， 在这些寄生虫中表达。然而，缺乏遗传学方法， 操纵和缺乏寄生虫材料已经限制并将继续 来限制对这些生物的研究。最近，有证据表明， 外源DNA序列可以被引入到丝虫的胚胎中 马来丝虫，采用基因枪方法和泛嗜性 逆转录病毒载体。本提案的总体目标是利用这些 克服传统遗传学缺乏所带来的局限性的系统 以及无法获得大量的寄生虫材料。具体 本提案的目的是： 1.利用基因枪瞬时转染系统， 控制B基因表达的重要因素。马来语。的参数 探索将包括鉴定瞬时表达的最小启动子， 在B中表达。马来人，5 '和3'序列的作用对 信息处理，以及添加合成内含子的效果。 2.建立泛嗜性逆转录病毒载体作为稳定的外源基因表达系统 在B中表达。马来胚胎 3.利用表达癌基因的泛嗜性逆转录病毒载体， 来自B的永生化细胞系。马来语。 4.确定转染的B中是否存在异源DNA。马来胚胎是 稳定遗传。

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.