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J Virol. 2010 Nov;84(22):11771-80. doi: 10.1128/JVI.01355-10. Epub 2010 Sep 15.

High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones.

Author information

1
Department of Microbiology,University of California, David Geffen School of Medicine, Los Angeles, CA 90095, USA.

Abstract

Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5' and the 3' vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.

PMID:
20844053
PMCID:
PMC2977897
DOI:
10.1128/JVI.01355-10
[Indexed for MEDLINE]
Free PMC Article

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