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Construction of a retrotransposition indicator sequence using a neomycin resistance-encoding gene containing a
D A Schwartz1, M W Dahm, L Bai
1Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston 29425.
Gene
|May 30, 1993
Summary
A Syrian hamster intron was added to a neo gene to create a novel selectable marker. This marker enables the identification of genomic integration sites after retrotransposition in mammalian cells.
Area of Science:
- Molecular biology
- Genetics
- Biotechnology
Background:
- Gene expression relies on proper RNA splicing.
- Selectable markers are crucial for genetic engineering and identifying successful gene insertions.
- Aminoglycoside phosphotransferase confers resistance to antibiotics like G418.
Purpose of the Study:
- To develop a novel selectable marker for identifying genomic integration sites.
- To engineer a gene construct that utilizes intron-mediated splicing for functional protein expression.
- To validate the efficacy of the new marker in mammalian cell systems.
Main Methods:
- Insertion of a Syrian hamster gene intron into a neo gene.
- Transfection of the engineered construct into mouse and hamster cells.
- Assessment of aminoglycoside phosphotransferase activity and G418 resistance.
Main Results:
- The engineered neo gene construct, containing the hamster intron, produced active aminoglycoside phosphotransferase upon mRNA splicing.
- The unspliced construct was inactive in Escherichia coli.
- The construct conferred G418 resistance in transfected mouse and hamster cells, indicating successful expression and function.
Conclusions:
- The intron-containing neo gene construct serves as an effective selectable marker.
- This marker facilitates the cloning and identification of genomic integration sites post-retrotransposition.
- The system offers a valuable tool for genetic manipulation and analysis in mammalian cells.