CHARACTERIZATION OF SFHR MODIFICATION OF GENOMIC HPRT

基因组 HPRT SFHR 修饰的表征

• 批准号: 8055672
• 负责人: Dieter C Gruenert
• 金额: $ 0.77万
• 依托单位: CALIFORNIA PACIFIC MED CTR RES INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055672
• 关键词: Affect; Aminopterin; Apoptosis; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Code; Culture Media; DNA; DNA Sequence; DNA analysis; Databases; Development; Effectiveness; Electroporation; Exons; Fibroblasts; Frequencies; Functional RNA; Gene Targeting; Genes; Genetic Polymorphism; Genetic Recombination; Genomics; Hereditary Disease; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Kinetics; Length; Lymphocyte; Mediating; Microinjections; Modification; Mutation; Play; Population; Proteins; Relative (related person); Resistance; Restriction fragment length polymorphism; Reverse Transcriptase Polymerase Chain Reaction; Role; Southern Blotting; System; TP53 gene; Testing; Therapeutic Intervention; Thymidine; base; human male; insertion/deletion mutation; lymphoblast; mutant; recombinational repair; research study

DESCRIPTION (provided by applicant): This project will test the general hypothesis that mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene can be used to characterize and optimize gene-targeting by small fragment homologous replacement (SFHR). Although SFHR has been successful, it has yet to be optimized or fully characterized in terms of the genomic modification of an endogenous eukaryotic gene. The HPRT gene is the ideal target for this project, because cells that contain a normal copy of the gene (HPRT+) can be readily distinguished from cells with a mutant copy of the gene (HPRT) by simply adding or omitting particular selective agents in the cell culture medium. As a result, it will be possible to determine if SFHR has successfully modified the HPRT locus through the isolation of clonal cell populations. Optimization and characterization of SFHR will be accomplished by evaluating different types of small DNA fragments (SDF) (single or double stranded varying in size), by evaluating how the target sequence affects SFHR-mediated gene modification (base substitutions, insertions, deletions, and frameshifts will be compared in different HPRT exons), and by evaluating the fate of the SDF in terms of the kinetics of SDF disappearance from the cell/nucleus and random insertion. A gene that has been implicated in DNA recombination, repair and replication is p53. Since some cell lines are p53+, while others are p53-, it will also be possible to evaluate the role that p53 plays in SFHR-mediated modification. The relative effectiveness of each type of SDF in modifying the HPRT gene will be determined by selection in hypoxanthine-aminopterin-thymidine (HAT) medium. In addition to this phenotypic selection for HAT resistance (HAT), PCR analysis of DNA will be used to screen for SFHR-mediated genomic alterations. Reverse transcriptase PCR (RT-PCR), Southern blot hybridization, sequencing and restriction length polymorphism (RFLP) analysis will be used for genotypic confirmation. Different mutations at the same or adjacent bases, frameshifts, and deletions will be analyzed. In addition, the affect of Alu sequences in the genomic target region on SFHR efficiency will be determined. Furthermore, random insertion and the degree to which it occurs will be assessed by Southern blot hybridization of the clonally selected cell populations along with the kinetics of SDF disappearance from the cell/nucleus will also be evaluated. Characterization and optimization of SFHR-mediated modification will be a significant step in the development of this approach as a therapeutic intervention for genetic diseases.

描述(由申请人提供)：该项目将测试次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HPRT)基因突变可用于通过小片段同源替换(SFHR)来表征和优化基因靶向的一般假设。虽然SFHR已经取得了成功，但在内源真核基因的基因组修饰方面，它还没有得到优化或完全表征。HPRT基因是这个项目的理想靶点，因为只需在细胞培养液中添加或省略特定的选择剂，就可以很容易地将含有该基因正常拷贝(HPRT+)的细胞与含有该基因突变拷贝(HPRT)的细胞区分开来。因此，有可能通过分离克隆细胞群体来确定SFHR是否成功地修改了HPRT基因座。SFHR的优化和鉴定将通过评估不同类型的小DNA片段(SDF)(单链或双链大小不等)、评估目标序列如何影响SFHR介导的基因修饰(将比较不同HPRT外显子中的碱基替换、插入、缺失和移码)以及根据SDF从细胞/核消失和随机插入的动力学来评估SDF的命运。与DNA重组、修复和复制有关的一个基因是p53。由于一些细胞系是P53+，而另一些是P53-，因此也有可能评估P53在SFHR介导的修饰中所起的作用。每种类型的SDF在修饰HPRT基因方面的相对有效性将通过在次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷(HAT)介质中的选择来确定。除了HAT抗性(HAT)的表型选择外，DNA的PCR分析将用于筛选SFHR介导的基因组改变。采用逆转录-聚合酶链式反应(RT-PCR)、Southern印迹杂交、测序和限制性内切酶长度多态性(RFLP)分析进行基因分型。将分析相同或相邻碱基、移码和缺失的不同突变。此外，还将确定基因组靶区中的Alu序列对SFHR效率的影响。此外，将通过克隆选择的细胞群体的Southern杂交来评估随机插入及其发生的程度，同时还将评估SDF从细胞/核中消失的动力学。SFHR介导的修饰的特征和优化将是将这一方法发展为遗传病治疗干预的重要一步。