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Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
Published on: September 23, 2010
RGB marking with lentiviral vectors for multicolor clonal cell tracking
Kristoffer Weber1, Michael Thomaschewski, Daniel Benten
1Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Germany.
Nature Protocols
|April 7, 2012
Summary
This study introduces RGB marking, a novel clonal cell tracking method using lentiviral vectors to create unique cell colors. This technique enables stable, long-term lineage tracing in various biological systems.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Lentiviral vectors enable clonal cell marking through unique insertion site patterns.
- Existing methods lack the capacity for high-throughput, multiplexed clonal analysis.
Purpose of the Study:
- To develop and validate a novel RGB marking system for precise clonal cell tracking.
- To enable simultaneous marking of individual cells with unique color codes for lineage tracing.
Main Methods:
- Simultaneous transduction of target cells with three lentiviral gene ontology (LeGO) vectors encoding red, green, and blue fluorescent proteins.
- Utilizing an additive color model to generate unique color identifiers for each cell.
- Assessing marking efficiency via flow cytometry and fluorescence microscopy.
Main Results:
- RGB marking generates a diverse spectrum of stable, unique colors in individual cells.
- Color codes remain stable through cell division, facilitating clonal tracking.
- The protocol allows for efficient marking within hours and assessment within days.
Conclusions:
- RGB marking provides a robust and efficient method for clonal cell tracking in vitro and in vivo.
- This technique offers a powerful tool for studying cell dynamics, development, and disease progression.
- The system's stability and multiplexing capability significantly advance lineage tracing methodologies.
