title: Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.
abstract: We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D005822:Genetic Vectors; D007739:L Cells; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics*; D012313:RNA / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D012641:Selection, Genetic; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes