Cloning of cDNAs from a mammalian expression library by a direct selection-amplification method.
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.
Chapman
L
L
Department of Medical Oncology, Rhode Island Hospital, Brown University, Providence 02903, USA.
Sang
J
J
Lin
S H
SH
Hixson
D C
DC
Thompson
N L
NL
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000818:Animals; D015703:Antigens, CD; D001483:Base Sequence; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary; D015723:Gene Library; D008322:Mammals; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D017931:DNA Primers; D018076:DNA, Complementary; D000251:Adenosine Triphosphatases