CHARACTERIZATION OF SFHR MODIFICATION OF GENOMIC HPRT

基因组 HPRT SFHR 修饰的表征

• 批准号: 7362411
• 负责人: Dieter C Gruenert
• 金额: $ 34.24万
• 依托单位: CALIFORNIA PACIFIC MED CTR RES INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7362411
• 关键词: Affect; Aminopterin; Apoptosis; 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; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Kinetics; Length; Lymphocyte; Mediating; Microinjections; Modification; Mutation; Play; Polymerase Chain Reaction; Population; Proteins; Range; 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; size

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）：本项目将测试次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HPRT）基因突变可用于表征和优化小片段同源置换（SFHR）基因靶向的一般假设。虽然SFHR已经取得了成功，但它还有待于在内源性真核基因的基因组修饰方面进行优化或充分表征。HPRT基因是该项目的理想靶标，因为通过简单地在细胞培养基中添加或省略特定的选择剂，可以容易地将含有该基因的正常拷贝（HPRT+）的细胞与具有该基因的突变拷贝（HPRT）的细胞区分开。因此，将有可能确定SFHR是否已通过分离克隆细胞群成功修饰HPRT基因座。SFHR的优化和表征将通过评估不同类型的小DNA片段（SDF）来完成。（单链或双链大小不同），通过评估靶序列如何影响SFHR介导的基因修饰（将在不同HPRT外显子中比较碱基置换、插入、缺失和移码），并通过根据SDF从细胞/细胞核中消失和随机插入的动力学来评估SDF的命运。参与DNA重组、修复和复制的基因是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。将通过在次黄嘌呤-氨基蝶呤-胸苷（HAT）培养基中选择来确定每种类型的SDF在修饰HPRT基因中的相对有效性。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。逆转录酶PCR（RT-PCR）、Southern印迹杂交、测序和限制性长度多态性（RFLP）分析将用于基因型确认。将分析相同或相邻碱基处的不同突变、移码和缺失。此外，将确定基因组靶区域中的Alu序列对SFHR效率的影响。此外，随机插入和其发生的程度将通过克隆选择的细胞群的Southern印迹杂交来评估，沿着还将评估SDF从细胞/细胞核消失的动力学。SFHR介导的修饰的表征和优化将是发展这种方法作为遗传疾病的治疗干预的重要一步。