Development of a retrovirus-based complementary DNA expression system for the cloning of tumor antigens.
A new retroviral system has been developed for the generation of a cDNA library and the functional cloning of tumor antigens. These retroviral vectors contain a cytomegalovirus promoter in the 5' long terminal repeat, an extended packaging signal for rapid production of high-titer retroviral particles, and many convenient cloning sites for cDNA library construction. The vesicular stomatitis virus G protein has been used to generate pseudotype retroviral particles to enable efficient viral infection. Using this system, viral titers in the range of 10(6) colony-forming units/ml could be generated routinely, and a high transduction efficiency in human primary cells, including fibroblasts, was achieved. In addition, a new procedure has been devised for screening a retrovirus-based cDNA library without a functional selection. The utility of this system was demonstrated by constructing a retrovirus-based cDNA library and re-isolating the NY-ESO-1 tumor antigen from a cDNA library using an antigen-specific CTL. This approach can facilitate the identification of novel tumor antigens recognized by T cells without knowledge of MHC class I restriction elements and is generally applicable for the isolation of any gene as long as a biological assay is available.
Surgery Branch, National Cancer Institute, Bethesda, Maryland 20892-1502, USA. rongfu@pop.nci.nih.gov
R F
RF
Wang
X
X
Wang
S L
SL
Johnston
G
G
Zeng
P F
PF
Robbins
S A
SA
Rosenberg
D016475:3T3 Cells; D000818:Animals; D000951:Antigens, Neoplasm / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D003001:Cloning, Molecular; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D012190:Retroviridae; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014162:Transfection
D000951:Antigens, Neoplasm; D016207:Cytokines; D018076:DNA, Complementary