A SIMPLE AND EFFICIENT PROCEDURE FOR GENERATING STABLE EXPRESSION LIBRARIES BY CDNA CLONING IN A RETROVIRAL VECTOR

cDNA expression cloning is a powerful method for the rescue and identi fication of genes that are able to confer a readily identifable phenot ype on specific cell types. Retroviral vectors provide several advanta ges over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express gen es in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libra ries by using a murine retroviral vector. Essentially, the method invo lves the directional cloning of cDNA into the retroviral vector and th e generation of pools of stable ecotropic virus producing cells from t his DNA. The cells so derived constitute the library, and the virus th ey yield is used to infect appropriate target cells for subsequent fun ctional screening. We have demonstrated the feasibility of this proced ure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate c DNAs for interleukin-3 and granulocyte-macrophage colony-stimulating f actor on the basis of the ability of these factors to confer autonomou s growth on the factor-dependent hemopoietic cell line FDC-P1. Moreove r, the frequency at which these factor-independent clones were isolate d approximated the frequency at which they were represented in the ori ginal plasmid library. These results suggest that expression cloning w ith retroviruses in a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.