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Title 

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

 

Repopulating hematopoietic stem cell clone을 대용량 시퀀싱으로 정량하는 방법

Authors 

S KimNamshin KimA P PressonD S AnS H MaoA C BonifacinoR E DonahueS A ChowI S Y Chen

Publisher 

American Society for Microbiology

Issue Date 

2010

Citation 

Journal of Virology, vol. 84, no. 22, pp. 11771-11780

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

Link 

http://dx.doi.org/10.1128/JVI.01355-10

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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