[Abstract]  [Full Text]  [PDF]

Sandra L. Fernandez¹, David W. Russell² and Peter J. Hurlin^3*

¹ Shriners Hospitals for Children. Department of Cell Developmental Biology, Oregon Health & Science Portland. OR 97201. USA.
² Department of Pediatrics, Division of Genetics and Developmental Medicine, University of Washington Seattle. WA 98195-7720. USA.
³ Department of Cell Developmental Biology, Oregon Health & Science Portland. OR 97201. USA. Phone: (503) 221 3438 Fax: (503) 221 3451 Email: pjh@shcc.org.

* To whom correspondence should be addressed: Peter J. Hurlin, Department of Cell Developmental Biology, Oregon Health & Science Portland. OR 97201. USA. Phone: (503) 221 3438. Fax: (503) 221 3451. Email: pjh@shcc.org

Biol. Proced. Online 2007;9:84-90. doi:10.1251/bpo136
Submitted: September 10, 2007; Accepted: November 19, 2007; Published: December 24, 2007.

Indexing terms: Dependovirus; Proto-Oncogene proteins c-myc.

Abbreviations: EGFP, enhanced green fluorescent protein; HFF, human foreskin fibroblast; rAAV, recombinant adeno-associated virus.

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.