Maintenance of insect cell cultures and generation of recombinant baculoviruses.
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.
Murphy
Cheryl Isaac
CI
Aquila Biopharmaceuticals, Worcester, Massachusetts, USA.
Piwnica-Worms
Helen
H
Grünwald
Stefan
S
Romanow
William G
WG
Francis
Nicole
N
Fan
Hua-Ying
HY
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*
D018076:DNA, Complementary; D004274:DNA, Recombinant; D004279:DNA, Viral; D007202:Indicators and Reagents