Related Experiment Videos
[Construction of eukaryotic expression vector using neomycin-resistance gene mutant as selectable marker]
Chuan Gao1, Xu-Dong Zhu, Xiao-Wei Zhou
1Institute of Pharmaceutical Chemistry, Beijing 102205, China. g.ch.chuan@263.net
Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology
|August 24, 2002
Summary
A modified neomycin-resistance gene enhances eukaryotic expression vectors, achieving high transfection rates and increased expression in CHO-K1 cells for genetic engineering applications.
Area of Science:
- Molecular Biology
- Genetic Engineering
Context:
- The neomycin-resistance gene (neo) is a standard selectable marker in eukaryotic expression vectors.
- It encodes neomycin phosphotransferase II (NPT II), providing resistance to antibiotics like G418 and kanamycin.
Purpose:
- To develop an improved selectable marker by site-directed mutagenesis of the neo gene.
- To construct an expression vector (pmDNA) utilizing the modified neo gene.
- To assess the efficiency of the modified vector in stable cell line development.
Summary:
- A mutant neo gene was created using site-directed mutagenesis.
- An expression vector, pmDNA, was constructed with this mutant neo gene as a selectable marker.
- The resulting expression plasmid, pmDNAluc+, containing a luciferase gene, was stably transfected into CHO-K1 cells.
Impact:
- The modified vector achieved a high expression-positive ratio of approximately 95% in transfected cells.
- A significantly higher ratio of high-expression colonies was observed compared to controls.
- This enhanced selectable marker system improves the efficiency of stable eukaryotic cell line construction.