Cellular barcoding tool for clonal analysis in the hematopoietic system

Alice Gerrits, Brad Dykstra, Olga J. Kalmykowa, Karin Klauke, Evgenia Verovskaya, Mathilde J. C. Broekhuis, Gerald de Haan*, Leonid V. Bystrykh

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

183 Citations (Scopus)

Abstract

Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important. (Blood. 2010;115(13):2610-2618)

Original languageEnglish
Pages (from-to)2610-2618
Number of pages9
JournalBlood
Volume115
Issue number13
DOIs
Publication statusPublished - 1-Apr-2010

Keywords

  • SELF-RENEWAL CAPACITY
  • STEM-CELLS
  • IN-VIVO
  • GENE-THERAPY
  • DEFICIENT MICE
  • TERM
  • DIFFERENTIATION
  • MOUSE
  • QUANTIFICATION
  • PRECURSORS

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