High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones = Repopulating hematopoietic stem cell clone을 대용량 시퀀싱으로 정량하는 방법

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Title
High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones = Repopulating hematopoietic stem cell clone을 대용량 시퀀싱으로 정량하는 방법
Author(s)
S Kim; Namshin Kim; A P Presson; D S An; S H Mao; A C Bonifacino; R E Donahue; S A Chow; I S Y Chen
Bibliographic Citation
Journal of Virology, vol. 84, no. 22, pp. 11771-11780
Publication Year
2010
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.
ISSN
0022-538X
Publisher
Amer Soc Microb
DOI
http://dx.doi.org/10.1128/JVI.01355-10
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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