RHODOPSIN GENE CORRECTION BY OLIGONUCLEOTIDE TARGETING

描述(改编自申请人摘要)：申请人建议 利用新方法对人类突变进行靶向基因校正 视紫红质基因与视网膜色素变性相关。站点定向 特定突变的纠正将由两个寡核苷酸完成 基础方法：三链形成寡核苷酸(TFOS)和RNA/DNA 嵌合寡核苷酸。这些基因检测方法的可行性 靶向将在两个层面上解决：(1)体外实验，使用 TFOS靶向视紫红质合成片段和质粒源片段 基因；和(2)修饰和未修饰的TFOS和RNA/DNA嵌合体 在人类细胞系中进行了测试，这些细胞系已经被设计为表达正常和 来自天然染色体的视紫红质基因的突变版本。 拟议研究的主要部分将是构建适当的 便于检测各种突变的细胞系。这将是 通过将绿色荧光蛋白(GFP)与视紫红质基因融合来完成。 这种结构将允许荧光激活的细胞分选用于 收集改变过的细胞。

DESCRIPTION (Adapted from applicant's abstract): The applicant proposes to utilize new approaches to target gene correction to mutations in the human rhodopsin gene associated with retinitis pigmentosa (RP). Site-directed correction of specific mutations will be accomplished by two oligonucleotide based approaches: triplex-forming oligonucleotides (TFOs) and RNA/DNA chimeric oligonucleotides. The feasibility of these approaches to gene targeting will be addressed at two levels: (1) in vitro experiments using TFOs to target both synthetic and plasmid-derived fragments of the rhodopsin gene; and (2) modified and unmodified TFOs and RNA/DNA chimeras will be tested in human cell lines that have been engineered to express normal and mutant versions of the rhodopsin gene from the native chromosomal locus. The major part of the proposed research will be to construct appropriate cell-lines to facilitate detection of a variety of mutations. This will be done by fusing the Green fluorescent Protein (GFP) to the rhodopsin gene. This construct will allow fluorescent-activated cell sorting to be used to collect altered cells.