Development of human gene reporter cell lines using rAAV mediated homologous recombination
- 24 December 2007
- journal article
- Published by Springer Science and Business Media LLC
- Vol. 9, 84-90
- https://doi.org/10.1251/bpo136
Abstract
Understanding mechanisms of gene regulation has broad therapeutic implications for human disease. Here we describe a novel method for generating human cell lines that serve as reporters of transcriptional activity. This method exploits the ability of recombinant adeno-associated virus to mediate the insertion of exogenous DNA sequences into specific genomic loci through homologous recombination. To overcome the severe size limitation of the rAAV for carrying exogenous DNA, an enhanced green fluorescent protein (EGFP)-Luciferase fusion gene was used as both a selectable marker and gene expression reporter. EGFP was used for selection of correctly targeted alleles by taking advantage of known regulatory conditions that activate transcription of specific genes. Using this method, we describe the generation of primary human fibroblasts that express EGFP-Luciferase under the control of the c-Myc oncogene.This publication has 26 references indexed in Scilit:
- Homologous recombination is required for AAV-mediated gene targetingNucleic Acids Research, 2006
- Precise hit: adeno-associated virus in gene targetingNature Reviews Microbiology, 2005
- Gene Targeting with Viral VectorsMolecular Therapy, 2005
- Gene Targeting by Adeno-Associated Virus Vectors Is Cell-Cycle DependentHuman Gene Therapy, 2005
- Targeted Correction of Single-Base-Pair Mutations with Adeno-Associated Virus Vectors under Nonselective ConditionsJournal of Virology, 2004
- Gene Targeting in Stem Cells from Individuals with Osteogenesis ImperfectaScience, 2004
- Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand BreaksMolecular and Cellular Biology, 2003
- Human cell knockoutsCurrent Opinion in Oncology, 2002
- MYC oncogenes and human neoplastic diseaseOncogene, 1999
- c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cellsNature, 1985