Uptake and intracellular fate of phage display vectors in mammalian cells.
Receptor-mediated endocytosis is exploited in experimental systems for selective delivery of genes and drugs into specific cells. To improve targeting efficiency of delivery vectors, we have used phage display technology to isolate novel ligands for endocytosed receptors. We show here that phage vectors internalized by mammalian cells via integrin-mediated endocytosis can be rescued by cell lysis and quantitated by infection of bacteria. Immediately following uptake, phage enter an intracellular compartment where they remain intact, with phage titer unaffected by the addition of chloroquine. Phage are then translocated to a second intracellular compartment in which they are inactivated and their titer affected by chloroquine. Immunofluorescence microscopy showed an association of the second compartment with supranuclear organelles. The ability to recover internalized phage in an infectious form from two distinctive intracellular compartments provides a means to select novel ligands from phage libraries for targeted delivery of macromolecules into mammalian cells.
Ivanenkov
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Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, OH 45267, USA.
Felici
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Menon
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D003090:Coliphages / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005822:Genetic Vectors / Q000187:drug effects / Q000493:pharmacokinetics*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism