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A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking
- Source :
- Molecular therapy : the journal of the American Society of Gene Therapy. 25(3)
- Publication Year :
- 2016
-
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.
- Subjects :
- 0301 basic medicine
Genetic Vectors
Gene Expression
Computational biology
Biology
DNA barcoding
Flow cytometry
03 medical and health sciences
Genes, Reporter
Transduction, Genetic
Drug Discovery
Gene Order
Genetics
medicine
Humans
Multiplex
Vector (molecular biology)
Codon
Molecular Biology
Pharmacology
Homeodomain Proteins
medicine.diagnostic_test
Competitive growth
Lentivirus
Gene Transfer Techniques
Reproducibility of Results
Cell Differentiation
Flow Cytometry
Hematopoietic Stem Cells
Luminescent Proteins
MicroRNAs
030104 developmental biology
Cell Tracking
Molecular Medicine
Original Article
Three generations
Subjects
Details
- ISSN :
- 15250024
- Volume :
- 25
- Issue :
- 3
- Database :
- OpenAIRE
- Journal :
- Molecular therapy : the journal of the American Society of Gene Therapy
- Accession number :
- edsair.doi.dedup.....9a1b002effe6dda14e241eaa0691a245