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


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.



enhanced green fluorescent protein


human foreskin fibroblast


recombinant adeno-associated virus


  1. Sedivy JM, Dutriaux A. Gene targeting and somatic cell genetics—a rebirth or a coming of age? Trends Genet. 1999;15:88–90.

    Article  PubMed  CAS  Google Scholar 

  2. Hirata R, Chamberlain J, Dong R, Russell DW. Targeted transgene insertion into human chromosomes by adenoassociated virus vectors. Nat. Biotech. 2002;20:735–738.

    Article  CAS  Google Scholar 

  3. Chamberlain JR, Schwarze U, Wang P, Hirata RK, Hankenson KD, Pace JM, Underwood RA, Song KM, Sussman M, Byers PH, Russell DW. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303:1198–1201.

    Article  PubMed  CAS  Google Scholar 

  4. Topaloglu O, Hurley PJ, Yildirim O, Civin CI, Bunz F Improved methods for the generation of human gene knockout and knockin cell lines. Nucleic Acids Res. 2005;33:e158

    Google Scholar 

  5. Bunz F. Human cell knockouts. Curr. Opin. Oncol. 2002;14:73–78.

    Article  PubMed  Google Scholar 

  6. Russell DW, and Hirata RK. Human gene targeting by viral vectors. Nat. Genet. 1998;18:325–330.

    Article  PubMed  CAS  Google Scholar 

  7. Vasileva A, Jessberger R. Precise hit: adeno-associated virus in gene targeting. Nat Rev. Microbiol. 2005;3:837–847.

    Article  PubMed  CAS  Google Scholar 

  8. Hirata RK, Russell DW. Design and packaging of adeno-associated virus gene targeting vectors. J. Virol. 2000;74:4612–4620.

    Article  PubMed  CAS  Google Scholar 

  9. Porteus MH, Cathomen T, Weitzman MD, Baltimore D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol. Cell. Biol. 2003;23:3558–3565.

    Article  PubMed  CAS  Google Scholar 

  10. Kohli M, Rago C, Lengauer C, Kinzler KW, Vogelstein B. Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res. 2004;32(1):e3.

    Google Scholar 

  11. Liu X, Yan Z, Luo M, Zak R, Li Z, Driskel RR, Huang Y, Tran N, Engelhardt JF. Targeted correction of single-base-pair mutations with adeno-associated virus vectors under nonselective conditions. J. Virol. 2004;78;4165–4175.

    Article  PubMed  CAS  Google Scholar 

  12. Hendrie PC, and Russell DW. Gene targeting with viral vectors. Mol. Ther. 2005;12:9–17.

    Article  PubMed  CAS  Google Scholar 

  13. Vasileva A, Linden RM, Jessberger R. Homologous recombination is required for AAV-mediated gene targeting. Nucleic Acids Res. 2006;34:3345–3360.

    Article  PubMed  CAS  Google Scholar 

  14. Grandori C, Cowley SM, James LP, Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell Dev. Biol. 2000;16:653–699.

    Article  PubMed  CAS  Google Scholar 

  15. Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene 1999;18:3004–3016.

    Article  PubMed  CAS  Google Scholar 

  16. Christoph F, Schmidt B, Schmitz-Drager BJ, Schulz WA. Over-expression and amplification of the c-myc gene in human urothelial carcinoma. Int. J. Cancer 1999;84:169–173.

    Article  PubMed  CAS  Google Scholar 

  17. Erisman MD, Rothberg PG, Diehl RE, Morse CC, Spandorfer JM, Astrin SM. Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene. Mol. Cell. Biol. 1985;5(8): 1969–1976.

    PubMed  CAS  Google Scholar 

  18. Spencer CA, Groudine M. Control of c-myc regulation in normal and neoplastic cells. Adv. Cancer Res. 1991;56:1–48.

    Article  PubMed  CAS  Google Scholar 

  19. Weber A, Liu J, Collins I, Levens D. TFIIH operates through an expanded proximal promoter to fine-tune c-myc expression. Mol. Cell. Biol. 2005;25:147–161.

    Article  PubMed  CAS  Google Scholar 

  20. Persson H, Gray HE, Godeau F. Growth-dependent synthesis of c-myc-encoded proteins: early stimulation by serum factors in synchronized mouse 3T3 cells. Mol. Cell. Biol. 1985;5:2903–2912.

    PubMed  CAS  Google Scholar 

  21. Rabbitts PH, Watson JV, Lamond A, Forster A, Stinson MA, Evan G, Fischer W, Atherton E, Sheppard R, Rabbitts TH. Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle. EMBO J. 1985;4:2009–2015.

    PubMed  CAS  Google Scholar 

  22. Dean M, Levine RA, Ran W, Kindy MS, Sonenshein GE, Campisi J. Regulation of c-myc transcription and mRNA abundance by serum growth factors and cell contact. J Biol. Chem. 1986;261:9161–9166.

    PubMed  CAS  Google Scholar 

  23. Ramsay G, Evan GI, Bishop JM. The protein encoded by the human proto-oncogene c-myc. Proc. Natl. Acad. Sci. U S A 1984;81:7742–7746.

    Article  PubMed  CAS  Google Scholar 

  24. Hann SR, Thompson CB, Eisenman RN. c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells. Nature 1985;314:366–369.

    Article  PubMed  CAS  Google Scholar 

  25. Veldwijk MR, Topaly J, Laufs S, Hengge UR, Wenz F, Zeller WJ, Fruehauf S. Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks. Mol Ther. 2002;6:272–278.

    Article  PubMed  CAS  Google Scholar 

  26. Trobridge G, Hirata RK, Russell DW. Gene targeting by adeno-associated virus vectors is cell-cycle dependent. Hum Gene Ther. 2005;16:522–526.

    Article  PubMed  CAS  Google Scholar 

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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 ( ), 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).

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Indexing terms

  • Dependovirus
  • Proto-Oncogene Proteins c-myc