Development of human gene reporter cell lines using rAAV mediated homologous recombination

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Author: Fernandez, SL
Author: Hurlin, PJ
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Methods Article

Development of human gene reporter cell lines using rAAV mediated homologous recombination

Sandra L. Fernandez¹, David W. Russell² and Peter J. Hurlin³^*

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.

Figure 1 Enlarged

Fig. 1:

Direct selection of targeted EGFP-Luciferase reporter gene insertion into the c-Myc locus using known c-Myc regulatory conditions. (A) Subconfluent primary human foreskin fibroblast cells are infected with rAAV targeting vectors when c-Myc is being actively transcribed or replicated in proliferative cells. Background reporter gene expression is silenced through density arrest and serum withdrawal. Reporter gene expression is induced by serum stimulation. Single EGFP-Luciferase positive cells are selected using FACS. Cloned cells are expanded and screened for gene targeting events by PCR, Southern blot, and sequencing. (B) FACS histograms are shown for wildtype and c-Myc rAAV targeted cells following cell cycle entry. FL1-A (EGFP) was plotted against FL2-A (no fluorofor) to center the parent population and allow for selection of the dim EGFP-Luciferase positive cells. The P2 region of the plot indicates the gating used for selection of the EGFP-Luciferase positive portion of the parent population.

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