Molecular cloning of receptor genes by transfection.
We have described a transfection method for the isolation of surface antigen genes which requires no mRNA or protein purification. Application of this technique results in the recovery of the entire gene in a single step since selection for expression of genomic DNA forms the basis of the procedure. Based on our results with the transferrin receptor gene and other systems, it is evident that large transcription units can be transferred and expressed in mouse L-cells. This size consideration represents a major advantage over the use of cosmid shuttle vectors for genomic DNA expression. In the case of genes which code for very long mRNAs this method may also have advantages over cDNA expression systems. Although we have described methods for FACS isolation of transfectants based on the binding of species specific antibodies to surface antigens, other methods of identifying transfected cells could be employed. For example, in combination with an appropriate assay, sib selection of recipient cells could be used to identify genes encoding secreted products. Ligand binding assays could be used for receptors which are not expressed on the host cell. Finally, the development of cDNA expression vectors which produce membrane-associated products would extend this methodology to genes not normally expressed at the cell surface.
McClelland
A
A
Kamarck
M E
ME
Ruddle
F H
FH
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005796:Genes; D007739:L Cells / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000235:genetics*; D014170:Transformation, Genetic
D000954:Antigens, Surface; D011956:Receptors, Cell Surface; D004247:DNA