UNIT 4.8 cDNA Expression Cloning in Mammalian Cells
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Neuroscience
How to Cite
Hoffman, B. J. 2001. cDNA Expression Cloning in Mammalian Cells. Current Protocols in Neuroscience. 3:4.8:4.8.1–4.8.23.
- Published Online: 1 MAY 2001
- Published Print: MAY 1998
This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate- or liposome-mediated transfection of mammalian cells, or virus infection and liposome-mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library-transformed E. coli grown in liquid culture medium or on antibiotic-containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion-coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter-based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library.