title: Maintenance of insect cell cultures and generation of recombinant baculoviruses.
abstract: This unit describes the maintenance and care of insect cell cultures as well as the generation, purification, and storage of recombinant baculoviruses. Procedures are included for maintenance and subculturing of insect cells and cotransfection of insect cells with linearized baculovirus DNA and recombinant transfer plasmid containing the gene of interest. In the event that the linearized virus is not available, wild-type baculovirus (AcMNPV) DNA may be used to produce recombinant baculoviruses. A procedure is also included for the generation of recombinant baculoviruses using a novel method, direct cloning, which eliminates the need to first clone the gene of interest into a baculoviral transfer vector. Preparation of baculovirus infection stocks from both monolayer and suspension cultures is also described. Finally, a protocol is given for a plaque assay to be used for determining the titer of baculoviral stocks as well as for selection of recombinants and plaque purification.
authors.affiliation: Aquila Biopharmaceuticals, Worcester, Massachusetts, USA.
authors.forename: Cheryl Isaac
authors.initials: CI
authors.lastname: Murphy
authors.forename: Helen
authors.initials: H
authors.lastname: Piwnica-Worms
authors.forename: Stefan
authors.initials: S
authors.lastname: Grünwald
authors.forename: William G
authors.initials: WG
authors.lastname: Romanow
authors.forename: Nicole
authors.initials: N
authors.lastname: Francis
authors.forename: Hua-Ying
authors.initials: HY
authors.lastname: Fan
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development* / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line / Q000378:metabolism / Q000821:virology; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D007202:Indicators and Reagents; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D014162:Transfection / Q000379:methods; D010948:Viral Plaque Assay; D014776:Virus Cultivation / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D004274:DNA, Recombinant; D004279:DNA, Viral; D007202:Indicators and Reagents