RESUMO
We describe a set of Moloney Murine Leukemia Virus (MoMLV)-based replication-defective retroviral vectors for delivery of the ecdysone-inducible system into mammalian cells. The vector pFB-ERV contains a tricistronic CMV expression cassette from which the ecdysone receptor proteins RXR and VgEcR are expressed, with the neo-resistance marker expressed as the third open reading frame (ORF). The inducible vector pCFB-EGSH contains an ecdysone-inducible expression cassette inserted between the viral LTRs in the antisense orientation relative to that for the viral promoter. Potential interference from the proviral 5' LTR is obviated due to a SIN deletion in the 3' LTR. When used together, induction ratios of over 1000-fold were achieved in NIH3T3 cells using firefly luciferase as a reporter.
Assuntos
Ecdisona/biossíntese , Ecdisona/genética , Marcação de Genes/métodos , Engenharia Genética/métodos , Rim/metabolismo , Vírus da Leucemia Murina de Moloney/genética , Transfecção/métodos , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Regulação da Expressão Gênica/genética , Humanos , Rim/embriologia , Proteínas Recombinantes/biossínteseRESUMO
Y box-binding protein 1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and of translation. In chicken embryo fibroblasts transformed by the oncoproteins P3k (phosphatidylinositol 3-kinase) or Akt, YB-1 is transcriptionally down-regulated. Expression of YB-1 from a retroviral vector induces a strong cellular resistance to transformation by P3k or Akt but does not affect sensitivity to transformation by other oncoproteins, such as Src, Jun, or Qin. The YB-1-expressing cells assume a tightly adherent, flat phenotype, with YB-1 localized in the cytoplasm, and show a greatly reduced saturation density. Both cap-dependent and cap-independent translation is inhibited in these cells, but the activity of Akt remains unaffected, suggesting that YB-1 functions downstream of Akt. A YB-1 protein with a loss-of-function mutation in the RNA-binding motif no longer binds to the mRNA cap structure, is localized in the cell nucleus, does not induce the flat cellular phenotype, and fails to interfere with P3k- or Akt-induced oncogenic transformation. This mutant also does not inhibit cap-dependent or cap-independent translation. These results suggest that YB-1 acts like a rapamycin mimic, inhibiting translational events that are required in phosphatidylinositol 3-kinase-driven oncogenic transformation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Fatores de Transcrição , Animais , Sequência de Bases , Sítios de Ligação , Divisão Celular , Células Cultivadas , Embrião de Galinha , DNA/genética , Expressão Gênica , Fatores de Transcrição NFI , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 de Ligação a Y-BoxRESUMO
Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.