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MDR1 bicistronic vectors: analysis of selection stringency, amplified gene expression, and vector stability in cell
S E Kane1, L Matsumoto, M Z Metz
1Department of Cell and Tumor Biology, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA. skane@coh.org
Biochemical Pharmacology
|September 12, 2001
Summary
The multidrug resistance-1 gene (MDR1) offers a stringent selection system for gene therapy. Higher drug pressure increases MDR1 expression, primarily through gene amplification, impacting cell population diversity.
Area of Science:
- Molecular Biology
- Gene Therapy
- Cell Biology
Background:
- The human multidrug resistance-1 gene (MDR1) is a key selectable marker in mammalian cells.
- MDR1 shows potential for gene therapy, protecting cells from chemotherapy and marking therapeutic gene overexpression.
- Sustained high MDR1 expression is crucial for conferring a survival advantage, but its selection system is stringent.
Purpose of the Study:
- To conduct a detailed molecular analysis comparing the stringency of MDR1 selection with the neo selectable marker.
- To investigate the impact of selection stringency on gene expression levels and mechanisms of adaptation.
- To evaluate the influence of selection stringency on the clonal diversity of transduced cell populations.
Main Methods:
- A bicistronic vector containing MDR1 and neo genes was introduced into NIH 3T3 and K562 cells.
- Cells were subjected to selection using colchicine (for MDR1) or G418 (for neo).
- Analysis included gene product expression, drug resistance, mRNA integrity, and vector DNA integration/copy number.
Main Results:
- Colchicine selection for MDR1 was found to be more stringent than G418 selection for neo.
- Increased selection pressure with colchicine correlated with elevated MDR1 gene expression.
- Gene amplification was identified as the primary mechanism for accommodating increased gene expression, even from single-copy integrations.
- Selection stringency significantly influenced the clonal diversity of transduced cell populations.
Conclusions:
- The stringency of MDR1 selection impacts gene expression and cellular adaptation mechanisms.
- Gene amplification plays a critical role in achieving high MDR1 expression under stringent selection.
- Understanding these dynamics is vital for optimizing MDR1's utility as a selectable marker and gene therapy tool, particularly in hematopoietic cells.
