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Replica filter screening technique to detect transfected cells expressing beta 2-adrenergic receptor
C A Machida1, J Bunzow, E Hanneman
1Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
DNA (Mary Ann Liebert, Inc.)
|July 1, 1989
Summary
A new screening assay identifies cells expressing beta 2-adrenergic receptors (beta 2-AR) using replica transfer. This method detects even single gene copies and low receptor numbers, aiding neurotransmitter receptor research.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Beta 2-adrenergic receptors (beta 2-AR) are crucial for cellular signaling.
- Efficiently identifying cells expressing specific receptors is vital for research.
Purpose of the Study:
- To develop a novel screening assay for identifying transfectants expressing beta 2-AR.
- To establish a sensitive method for detecting low levels of receptor expression.
Main Methods:
- Utilized a replica transfer technique for screening.
- Cotransfected beta 2-AR genes with neomycin resistance plasmids into mouse L cells.
- Screened colonies using radioligand filter binding assay with [125I]iodohydroxybenzylpindolol.
Main Results:
- Successfully identified transfectants expressing beta 2-AR.
- The assay detected cells with a single beta 2-AR gene copy.
- Identified cells expressing as few as 4,000 beta 2-receptors per cell.
Conclusions:
- The developed replica transfer assay is a sensitive tool for identifying cells expressing beta 2-AR.
- This method can be used for molecular cloning of neurotransmitter receptor genes.
- Useful for measuring transfection efficiencies and receptor gene expression.