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Journal of Virology, March 2008, p. 2448-2455, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.00584-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Multiple Displacement Amplification Enables Large-Scale Clonal Analysis following Retroviral Gene Therapy{triangledown}

S. Bleier,1,{dagger} P. Maier,2,{dagger} H. Allgayer,3 F. Wenz,2 W. J. Zeller,1 S. Fruehauf,4 and S. Laufs3*

Research Program Innovative Cancer Diagnostics and Therapy, German Cancer Research Center, Heidelberg, Germany,1 Department of Radiation Oncology, Mannheim Medical Center, University of Heidelberg, Heidelberg, Germany,2 Department of Experimental Surgery and Molecular Oncology of Solid Tumors, Medical Faculty Mannheim, University of Heidelberg, and German Cancer Research Center (DKFZ) Heidelberg, Mannheim, Germany,3 Center for Tumor Diagnostics and Therapy, Paracelsus Klinik, Osnabrueck, Germany4

Received 20 March 2007/ Accepted 13 December 2007

Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.

* Corresponding author. Mailing address: Molecular Oncology of Solid Tumors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Phone: 49-6221-421710. Fax: 49-6221-421719. E-mail: s.laufs{at}dkfz.de

{triangledown} Published ahead of print on 12 December 2007.

{dagger} These authors contributed equally to this work.

Journal of Virology, March 2008, p. 2448-2455, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.00584-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Moalic, Y., Felix, H., Takeuchi, Y., Jestin, A., Blanchard, Y. (2009). Genome Areas with High Gene Density and CpG Island Neighborhood Strongly Attract Porcine Endogenous Retrovirus for Integration and Favor the Formation of Hot Spots. J. Virol. 83: 1920-1929 [Abstract] [Full Text]  


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