Related Experiment Videos
Improved expression cloning using reporter genes and Epstein-Barr virus ori-containing vectors
E S Shen1, G M Cooke, R A Horlick
1DuPont Merck Pharmaceutical Co., Experimental Station, Wilmington, DE 19880-0400, USA.
Gene
|April 24, 1995
Summary
The 293EBNA cell line significantly enhances expression cloning efficiency compared to T-antigen cell lines. It shows higher reporter gene expression, enabling more sensitive detection for applications like angiotensin II type-1 receptor cloning.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- SV40 and Epstein-Barr virus (EBV) ori-containing plasmids are used for gene expression studies.
- Cell lines constitutively expressing T-antigen (TSA201, COS7) or EBV nuclear antigen 1 (EBNA1, 293EBNA) support plasmid replication.
Purpose of the Study:
- To compare the expression cloning capabilities of 293EBNA cells with T-antigen-based cell lines (COS7, TSA201).
- To evaluate reporter gene expression levels and detection sensitivity for specific genes.
Main Methods:
- Transient transfection of reporter gene constructs into 293EBNA, COS7, and TSA201 cell lines.
- Measurement of beta-galactosidase (beta Gal) and angiotensin II type-1 receptor (AT1) expression levels.
- Assessment of detection limits using serial dilutions of reporter plasmids and a NaOH 'scrape' method.
Main Results:
- 293EBNA cells exhibited significantly higher expression of beta Gal (>25-fold) and AT1 (70-100-fold) compared to COS7 and TSA201 cells.
- Expression cloning sensitivity was markedly improved in 293EBNA cells, allowing reporter plasmid detection at 80,000-fold dilutions versus 1000-fold for others.
- The NaOH 'scrape' method proved effective for reporter gene detection.
Conclusions:
- 293EBNA cells represent a superior system for expression cloning compared to traditional T-antigen-based cell lines.
- The enhanced sensitivity and expression levels in 293EBNA cells facilitate more efficient gene discovery and characterization.