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Methods Mol Biol. 2014;1185:345-60. doi: 10.1007/978-1-4939-1133-2_23.

Barcoded vector libraries and retroviral or lentiviral barcoding of hematopoietic stem cells.

Author information

1
Laboratory of Ageing Biology and Stem Cells, European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Antoninus Deusinglaan 1, Building 3226, 9713 AV, Groningen, The Netherlands, l.bystrykh@umcg.nl.

Abstract

Cellular barcoding is a relatively recent technique aimed at clonal analysis of a proliferating cell population of any kind. The method was shown to be particularly successful in monitoring clonal contributions of hematopoietic stem cells (HSCs). An essential step of the method is retroviral or lentiviral labeling of the hematopoietic cells. The unique feature of the method is the generation of a vector library containing specific artificial DNA tags, generally known as barcodes. The library must satisfy multiple essential requirements. Importantly, considering the number of possible variations within the barcode sequence, the actual size of the barcoded vector library, and the number of clonogenic (stem) cells in the given experiment should be in ratios far from saturation. Excessive bias in barcodes frequencies must be avoided, and the library size must be assessed prior to the sequencing analysis. The final sequencing results must undergo statistical filtering. If all requirements are met, the method ensures profound sensitivity and accuracy for monitoring of the clonal fluctuations in a wide range of biological experiments.

PMID:
25062640
DOI:
10.1007/978-1-4939-1133-2_23
[Indexed for MEDLINE]

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