RHODOPSIN GENE CORRECTION BY OLIGONUCLEOTIDE TARGETING

描述（改编自申请人摘要）：申请人拟 利用新的方法对人类中的突变进行靶向基因校正， 视紫红质基因与视网膜色素变性（RP）相关。 定点 特异性突变的校正将通过两个寡核苷酸 三链体形成寡核苷酸（TFOs）和RNA/DNA 嵌合寡核苷酸。 这些方法的可行性基因 靶向将在两个水平上解决：（1）体外实验， 靶向视紫红质的合成片段和质粒衍生片段的TFO 基因;和（2）修饰和未修饰的TFO和RNA/DNA嵌合体将被 在人类细胞系中进行了测试，这些细胞系已经被工程化以表达正常和 来自天然染色体基因座的视紫红质基因的突变形式。 建议研究的主要部分将是建立适当的 细胞系，以便于检测各种突变。 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。 该构建体将允许使用荧光激活的细胞分选， 收集变异细胞

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.