title: A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking.
abstract: Retroviral integration site analysis and barcoding have been instrumental for multiplex clonal fate mapping, although their use imposes an inherent delay between sample acquisition and data analysis. Monitoring of multiple cell populations in real time would be advantageous, but multiplex assays compatible with flow cytometric tracking of competitive growth behavior are currently limited. We here describe the development and initial validation of three generations of lentiviral fluorescent genetic barcoding (FGB) systems that allow the creation of 26, 14, or 6 unique labels. Color-coded populations could be tracked in multiplex in vitro assays for up to 28 days by flow cytometry using all three vector systems. Those involving lower levels of multiplexing eased color-code generation and the reliability of vector expression and enabled functional in vitro and in vivo studies. In proof-of-principle experiments, FGB vectors facilitated in vitro multiplex screening of microRNA (miRNA)-induced growth advantages, as well as the in vivo recovery of color-coded progeny of murine and human hematopoietic stem cells. This novel series of FGB vectors provides new tools for assessing comparative growth properties in in vitro and in vivo multiplexing experiments, while simultaneously allowing for a reduction in sample numbers by up to 26-fold.
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany. Electronic address: tmaetzig@bccrc.ca.
authors.forename: Tobias
authors.initials: T
authors.lastname: Maetzig
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Jens
authors.initials: J
authors.lastname: Ruschmann
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Courteney K
authors.initials: CK
authors.lastname: Lai
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Mor
authors.initials: M
authors.lastname: Ngom
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Suzan
authors.initials: S
authors.lastname: Imren
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Patricia
authors.initials: P
authors.lastname: Rosten
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Gudmundur L
authors.initials: GL
authors.lastname: Norddahl
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Niklas
authors.initials: N
authors.lastname: von Krosigk
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Lea
authors.initials: L
authors.lastname: Sanchez Milde
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Christopher
authors.initials: C
authors.lastname: May
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Selich
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Rothe
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Ishpreet
authors.initials: I
authors.lastname: Dhillon
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada; Department of Medicine, University of British Columbia, Vancouver, BC V5Z 1M9, Canada.
authors.forename: R Keith
authors.initials: RK
authors.lastname: Humphries
mesh_terms: D002454:Cell Differentiation; D058948:Cell Tracking / Q000379:methods*; D003062:Codon; D005434:Flow Cytometry; D015870:Gene Expression; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics; D015203:Reproducibility of Results; D014161:Transduction, Genetic
chemical_list: D003062:Codon; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D035683:MicroRNAs; C097899:homeobox protein HOXA9
keywords: fluorescent genetic barcoding; hematopoietic stem cells; lentiviral gene transfer; multiplexing