title: Site-specific recombinatorics: in situ cellular barcoding with the Cre Lox system.
abstract: Cellular barcoding is a recently developed biotechnology tool that enables the familial identification of progeny of individual cells in vivo. In immunology, it has been used to track the burst-sizes of multiple distinct responding T cells over several adaptive immune responses. In the study of hematopoiesis, it revealed fate heterogeneity amongst phenotypically identical multipotent cells. Most existing approaches rely on ex vivo viral transduction of cells with barcodes followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment such as those inside solid tissues.
  
  
  
  With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. We identify the cassette whose provably maximal code diversity is several orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches, exceeding the number of lymphocytes or hematopoietic progenitor cells in mice.
  
  
  
  Its high diversity and in situ applicability, make the proposed Cre Lox based tagging system suitable for whole tissue or even whole animal barcoding. Moreover, it can be built using established technology.
authors.affiliation: Hamilton Institute, Maynooth University, Maynooth, Ireland.
authors.forename: Tom S
authors.initials: TS
authors.lastname: Weber
authors.affiliation: University of Strathclyde, Glasgow, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Dukes
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research & The University of Melbourne, Parkville, Melbourne, Australia.
authors.forename: Denise C
authors.initials: DC
authors.lastname: Miles
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research & The University of Melbourne, Parkville, Melbourne, Australia.
authors.forename: Stefan P
authors.initials: SP
authors.lastname: Glaser
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research & The University of Melbourne, Parkville, Melbourne, Australia.
authors.forename: Shalin H
authors.initials: SH
authors.lastname: Naik
authors.affiliation: Hamilton Institute, Maynooth University, Maynooth, Ireland. ken.duffy@nuim.ie.
authors.forename: Ken R
authors.initials: KR
authors.lastname: Duffy
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D014644:Genetic Variation; D019426:Integrases / Q000378:metabolism*; D055029:Inverted Repeat Sequences / Q000235:genetics; D051379:Mice; D011995:Recombination, Genetic
chemical_list: C045073:Cre recombinase; D019426:Integrases
keywords: Cell fate tracking; Cellular barcoding; Combinatorial explosion; Cre lox system; DNA stochastic programme