A sensitive and quantitative polymerase chain reaction-based cell free in vitro non-homologous end joining assay for hematopoietic stem cells

PLoS One. 2012;7(3):e33499. doi: 10.1371/journal.pone.0033499. Epub 2012 Mar 20.

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 µg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34(+) 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 contrast, mouse quiescent HSCs (Pyronin-Y(low) LKS(+) cells) and cycling HSCs (Pyronin-Y(hi) LKS(+) cells) repaired the damage more efficiently than HPCs (LKS(-) cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Assay*
  • Blotting, Western
  • Bone Marrow / metabolism
  • Cells, Cultured
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair*
  • DNA Helicases / metabolism
  • DNA-Activated Protein Kinase / metabolism
  • Flow Cytometry
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • In Vitro Techniques
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Nuclear Proteins / metabolism
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Nuclear Proteins
  • RNA, Messenger
  • DNA-Activated Protein Kinase
  • DNA Helicases