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Method for Efficient Transduction of Cancer Stem Cells
Kiera Walker1, Anita Hjelmeland1
1Department of Cell, Developmental and Integrative Biology; University of Alabama at Birmingham; Birmingham, Alabama 35294; USA.
Abstract:
Ectopic gene expression through introduction of cDNA and gene silencing by RNA interference each facilitate the elucidation of molecular pathways in both normal and pathologic states. As transfection efficiency in some primary and established cells is low, lentivirus based expression systems with high infection rates can improve experimental design. For example, glioblastoma cells and particularly the cancer stem cell (CSC) fraction can be difficult to transfect but are amenable to viral infection. Greater utilization of lentivirus for expression of cDNA and shRNA in CSCs may be limited due to technical challenges, including elimination of pro-differentiating fetal bovine serum (FBS). We therefore generated a subline of 293Ts that can proliferate and efficiently produce virus in CSC media, designated CSC293Ts. We provide detailed protocols for the generation of CSC293Ts and for the production of lentivirus for CSC infection using glioblastoma as a model. Our data demonstrate that serum free media from CSC293Ts consistently produces greater than 80% infection rates without virus concentration. We believe that the detailed protocols provided here can be adapted for multiple cell types for broad utility.
Insights
This study introduces CSC293Ts, a cell line that efficiently produces lentivirus in serum-free media. This overcomes challenges in gene expression and silencing in cancer stem cells (CSCs), improving molecular pathway research.
Area of Science:
- Molecular Biology
- Virology
- Cancer Research
Background:
- Ectopic gene expression and gene silencing are crucial for studying molecular pathways.
- Lentivirus-based systems offer high infection rates, beneficial for cells with low transfection efficiency, such as glioblastoma cancer stem cells (CSCs).
- Existing methods face challenges, including the need to eliminate fetal bovine serum (FBS), which can induce differentiation.
Purpose of the Study:
- To develop a lentivirus production system that functions effectively in serum-free media suitable for CSCs.
- To overcome technical limitations hindering the use of lentivirus for gene manipulation in CSCs.
- To provide protocols for generating and utilizing this system for glioblastoma research.
Main Methods:
- Generation of a 293T cell subline (CSC293Ts) capable of viral production in CSC media.
- Detailed protocols for CSC293T generation and lentivirus production.
- Infection of glioblastoma cells using lentivirus produced in serum-free conditions.
Main Results:
- CSC293Ts efficiently produce lentivirus in serum-free media.
- Achieved infection rates exceeding 80% in glioblastoma cells without the need for virus concentration.
- Demonstrated the utility of the system in a glioblastoma model.
Conclusions:
- The CSC293T cell line and associated protocols facilitate high-efficiency lentiviral gene transfer in CSCs using serum-free conditions.
- This system simplifies experimental design and broadens the applicability of lentivirus technology for CSC research.
- The provided protocols are adaptable for various cell types, offering wide utility.
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