Transforming lepidopteran insect cells for continuous recombinant protein expression.
The baculovirus expression vector system is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly because of the adverse effects of baculovirus infection on the host insect cell secretory pathway. An alternative approach to producing poorly expressed proteins is to transform insect cells with the gene of interest under the control of promoters that are constitutively active in uninfected cells, thereby making cell lines that continuously express recombinant protein. This chapter provides an overview of the methods and considerations for making stably transformed lepidopteran cells. Techniques for the insertion of genes into continuous expression vectors, transfection of cells, and the selection and isolation of stably transformed Sf-9 clones by either colony formation or end-point dilution are described in detail.
Harrison
Robert L
RL
USDA/ARS Insect Biocontrol Laboratory, Beltsville, MD, USA.
Jarvis
Donald L
DL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007915:Lepidoptera / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D011994:Recombinant Proteins