CHARACTERIZATION OF SFHR MODIFICATION OF GENOMIC HPRT

项目描述（由申请人提供）：本项目将验证关于次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）基因突变可用于表征和优化小片段同源替代（SFHR）基因靶向的一般假设。虽然SFHR已经取得了成功，但它还没有在内源性真核基因的基因组修饰方面得到优化或充分表征。HPRT基因是这个项目的理想目标，因为含有正常基因拷贝（HPRT+）的细胞可以很容易地与含有突变基因拷贝（HPRT）的细胞区分，只需在细胞培养基中添加或省略特定的选择性试剂。因此，通过克隆细胞群体的分离，将有可能确定SFHR是否成功地修饰了HPRT位点。SFHR的优化和表征将通过评估不同类型的小DNA片段（SDF）（大小不一的单链或双链），通过评估靶序列如何影响SFHR介导的基因修饰（碱基替换、插入、缺失和帧移将在不同的HPRT外显子中进行比较），以及根据SDF从细胞/细胞核中消失和随机插入的动力学来评估SDF的命运来完成。一个与DNA重组、修复和复制有关的基因是p53。由于一些细胞系是p53+，而另一些细胞系是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修饰HPRT基因的相对有效性将通过在次黄嘌呤-氨基蝶呤-胸腺嘧啶（HAT）培养基中的选择来确定。除了对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 blot杂交、测序和限制性内切长度多态性（RFLP）分析将用于基因型确认。将分析相同或相邻碱基的不同突变、帧移和缺失。此外，还将确定基因组靶区的Alu序列对SFHR效率的影响。此外，随机插入及其发生的程度将通过克隆选择的细胞群体的Southern blot杂交来评估，同时也将评估SDF从细胞/细胞核中消失的动力学。表征和优化sfhr介导的修饰将是该方法作为遗传疾病治疗干预手段发展的重要一步。

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.