Localization of the cystic fibrosis gene product in a transfected Sf9 insect cell line

Abstract

The cystic fibrosis gene product (CFTR) is thought to be either directly or indirectly involved with Cl- conductance in epithelial cells in which it is produced. Utilizing the Baculovirus expression vector system, CFTR has been expressed in Sf9 insect cells which in turn display a regulated Cl- conductance employing the same techniques as those used to demonstrate Cl- conductance in epithelial cells. This was not observed in mock-infected Bgalactosidase producing or non-infected Sf9 cells.Two cell lines, one infected with CFTR expressing recombinant baculovirus and the other infected with B-galactosidase expressing recombinant baculovirus were fixed in 2% paraformaldehyde and 0.001% glutaraldehyde in 0.1 M HEPES buffer, PH7.2 for 2 hours. They were then rinsed several times with buffer, infiltrated with 2% agarose and infused with 2.3 M sucrose for at least 24 hours. The samples were then frozen by rapid immersion in liquid nitrogen cooled FREON 22 and ultrathin cryosections cut with a cryoultramicrotome and immunogold labelled with purified monclonal antibodies against CFTR (1) using the method described by Griffiths.