Cellular barcoding tool for clonal analysis in the hematopoietic system.
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.
Gerrits
Alice
A
Department of Cell Biology, Section Stem Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Dykstra
Brad
B
Kalmykowa
Olga J
OJ
Klauke
Karin
K
Verovskaya
Evgenia
E
Broekhuis
Mathilde J C
MJ
de Haan
Gerald
G
Bystrykh
Leonid V
LV
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
D004274:DNA, Recombinant; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides