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

Biological Procedures Online volume 9, pages 84–90 (2007)Cite this article

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.

Abbreviations

EGFP:

enhanced green fluorescent protein

HFF:

human foreskin fibroblast

rAAV:

recombinant adeno-associated virus

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Authors and Affiliations

  1. Shriners Hospitals for Children, Oregon Health & Science Portland, 97201, OR, USA

    Sandra L. Fernandez & Peter J. Hurlin

  2. Department of Cell Developmental Biology, Oregon Health & Science Portland, 97201, OR, USA

    Sandra L. Fernandez & Peter J. Hurlin

  3. Department of Pediatrics, Division of Genetics and Developmental Medicine, University of Washington Seattle, 98195-7720, WA, USA

    David W. Russell

Authors
  1. Sandra L. Fernandez
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Correspondence to Peter J. Hurlin.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fernandez, S.L., Russell, D.W. & Hurlin, P.J. Development of human gene reporter cell lines using rAAV mediated homologous recombination. Biol. Proced. Online 9, 84–90 (2007). https://doiorg.publicaciones.saludcastillayleon.es/10.1251/bpo136

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1251/bpo136

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Biological Procedures Online

ISSN: 1480-9222

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