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. ? ? ?
| de Semir, D; Maurisse, R; Du, F et al. (2012) Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer. Cell Tissue Res 347:357-67 |
| Sargent, R Geoffrey; Kim, Soya; Gruenert, Dieter C (2011) Oligo/polynucleotide-based gene modification: strategies and therapeutic potential. Oligonucleotides 21:55-75 |
| Maurisse, Rosalie; De Semir, David; Emamekhoo, Hamid et al. (2010) Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol 10:9 |
| Bedayat, Babak; Abdolmohamadi, Alireza; Ye, Lin et al. (2010) Sequence-specific correction of genomic hypoxanthine-guanine phosphoribosyl transferase mutations in lymphoblasts by small fragment homologous replacement. Oligonucleotides 20:7-16 |
