title: Cellular barcoding tool for clonal analysis in the hematopoietic system.
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.
authors.affiliation: Department of Cell Biology, Section Stem Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Alice
authors.initials: A
authors.lastname: Gerrits
authors.forename: Brad
authors.initials: B
authors.lastname: Dykstra
authors.forename: Olga J
authors.initials: OJ
authors.lastname: Kalmykowa
authors.forename: Karin
authors.initials: K
authors.lastname: Klauke
authors.forename: Evgenia
authors.initials: E
authors.lastname: Verovskaya
authors.forename: Mathilde J C
authors.initials: MJ
authors.lastname: Broekhuis
authors.forename: Gerald
authors.initials: G
authors.lastname: de Haan
authors.forename: Leonid V
authors.initials: LV
authors.lastname: Bystrykh
mesh_terms: D000818:Animals; D016010:Binomial Distribution; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods; D002999:Clone Cells / Q000737:chemistry*; D004274:DNA, Recombinant / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D005819:Genetic Markers; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000032:analysis / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry* / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009838:Oligodeoxyribonucleotides / Q000032:analysis*; D012190:Retroviridae / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D019076:Transgenes; D016662:Virus Integration
chemical_list: D004274:DNA, Recombinant; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides