Abstract
A sensitive method was devised for cloning cDNAs from a mammalian expression library based on single-cell detection and selection of transfected cells. The method is applicable for cloning cell-surface or cytoplasmic proteins for which a detection assay, such as immunofluorescence or immunohistochemical reactivity, exists. The widely used eukaryotic expression vector cdm8 is utilized, and the method is demonstrated using the gene for C-CAM, a liver glycoprotein adhesion molecule. After transfection, cells are plated out on a collagen gel substrate to allow retrieval of selected cells. Simultaneous fixation and permeabilization with acetone permits immunological and histochemical detection of cell-surface and cytoplasmic proteins without loss of plasmid vector. Inserts of interest are recovered by PCR with vector primers.
Original language | English (US) |
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Pages (from-to) | 77-83 |
Number of pages | 7 |
Journal | Applied Biochemistry and Biotechnology - Part B Molecular Biotechnology |
Volume | 5 |
Issue number | 2 |
DOIs | |
State | Published - 1996 |
Keywords
- Expression cloning
- Immunoselection
- cDNA
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Biochemistry
- Applied Microbiology and Biotechnology
- Molecular Biology