Functional genomics in cancer research: identification of target genes of the Epstein-Barr virus nuclear antigen 2 by subtractive cDNA cloning and high-throughput differential screening using high-density agarose gels.
In the past, the identification and isolation of phenotype-associated genes was a difficult and time-consuming task. However, recent improvements of methods that are designed to isolate differentially expressed genes have remarkably speeded up the process of target gene isolation. The ultimate goal of functional genomics is to apply these technologies to clone phenotype-associated genes irrespective of the availability of probes (e.g., antibodies) and an intimate knowledge of biological background. We demonstrate the use of a novel subtractive cDNA cloning approach for the isolation and characterization of target genes of the Epstein-Barr virus nuclear antigen 2 (EBNA2). Two different subtractive cDNA libraries specific for two different time periods following activation of a conditional estrogen receptor/EBNA2 (ER/EBNA2) fusion protein were generated. Comparison of the two libraries by cross-hybridization experiments allowed the differentiation between direct and indirect target genes of EBNA2 and led to the identification of a novel direct target gene of EBNA2.
Kaiser
C
C
GSF Forschungszentrum für Umwelt und Gesundheit, Institute of Clinical Molecular Biology and Tumor Genetics, München, Germany.
von Stein
O
O
Laux
G
G
Hoffmann
M
M
D003001:Cloning, Molecular; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000378:metabolism; D005786:Gene Expression Regulation; D015698:Genomic Library; D006801:Humans; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000235:genetics
D018076:DNA, Complementary; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D004967:Estrogens; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins