Unit

UNIT 4.8 cDNA Expression Cloning in Mammalian Cells

  1. Beth J. Hoffman

Published Online: 1 MAY 2001

DOI: 10.1002/0471142301.ns0408s03

Current Protocols in Neuroscience

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.

Author Information

  1. National Institute of Mental Health, Bethesda, Maryland

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAY 1998

Abstract

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.