原创力文档a sensitive and quantitative polymerase chain reaction-based cell free in vitro non-homologous end joining assay for hematopoietic stem cells一个敏感和定量聚合酶链反应细胞体外自由异源端加入造血干细胞的鉴定.pdfVIP
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a sensitive and quantitative polymerase chain reaction-based cell free in vitro non-homologous end joining assay for hematopoietic stem cells一个敏感和定量聚合酶链反应细胞体外自由异源端加入造血干细胞的鉴定
A Sensitive and Quantitative Polymerase Chain Reaction-
Based Cell Free In Vitro Non-Homologous End Joining
Assay for Hematopoietic Stem Cells
1 1 2 2 1
Lijian Shao , Wei Feng , Kyung-Jong Lee , Benjamin P. C. Chen , Daohong Zhou *
1 Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America,
2 Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
Abstract
Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for
the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends
on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and
lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a
sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ) assay using linearized plasmids as the
substrates and quantitative polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via
NHEJ in less than 1 mg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34+
+ 2
hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34 CD38 cells) are less proficient in
the repair of DSBs by NHEJ than HPCs (CD34+CD38+ cells). In c
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