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 identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes 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 libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.
Rayner
J R
JR
Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, Australia.
Gonda
T J
TJ
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism*; D015723:Gene Library; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D016178:Granulocyte-Macrophage Colony-Stimulating Factor