New experimental approaches in retrovirus-mediated expression screening.
We have established an expression screening system that utilizes retrovirus-mediated gene transfer. The system is based on a high-efficiency packaging cell line BOSC23 and retrovirus vectors pBabeX and pMX which are suitable for cDNA library construction. The combination of BOSC23 packaging cells and the pBabeX and pMX retrovirus vectors produces high-titer recombinant retroviruses, which are able to cover large complexities of cDNA libraries. These retroviruses gave 100% infection efficiency in NIH3T3 cells and 10-100% infection efficiency in various hemopoietic cell lines. In contrast to the conventional mammalian expression cloning system where it is required to transduce cDNA transiently into particular cell types such as COS cells, the retrovirus-mediated expression cloning method allows stable transduction of cDNAs into a wide variety of cell types. This method, therefore, makes it possible to select cells expressing the desired cDNA by various functional assays. In addition, when combined with PCR-driven random mutagenesis, this system is also useful in searching for mutations of various molecules which will result in functional alterations. Other potential applications will also be discussed.
Kitamura
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Department of Hemopoietic Factors, University of Tokyo, Japan.
D016475:3T3 Cells; D000818:Animals; D002411:Cation Exchange Resins; D002460:Cell Line; D002469:Cell Separation; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008055:Lipids; D051379:Mice; D016133:Polymerase Chain Reaction; D018121:Receptors, Cytokine / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
D002411:Cation Exchange Resins; D018076:DNA, Complementary; D008055:Lipids; C086724:Lipofectamine; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins