Unit

UNIT 16.10 Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses

  1. Cheryl Isaac Murphy1,
  2. Helen Piwnica-Worms2,
  3. Stefan Grünwald3,
  4. William G. Romanow4,
  5. Nicole Francis5,
  6. Hua-Ying Fan5

Published Online: 1 FEB 2004

DOI: 10.1002/0471142727.mb1610s65

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Murphy, C. I., Piwnica-Worms, H., Grünwald, S., Romanow, W. G., Francis, N. and Fan, H.-Y. 2004. Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses. Current Protocols in Molecular Biology. 65:II:16.10:16.10.1–16.10.19.

Author Information

  1. 1

    Aquila Biopharmaceuticals, Worcester, Massachusetts

  2. 2

    Washington University School of Medicine, St. Louis, Missouri

  3. 3

    Pharmingen, San Diego, California

  4. 4

    Corning Costar, Portsmouth, New Hampshire

  5. 5

    Massachusetts General Hospital, Boston, Massachusetts

Publication History

  1. Published Online: 1 FEB 2004
  2. Published Print: JAN 2004

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.