Retrovirus-mediated gene transfer of beta-nerve growth factor into mouse pituitary line AtT-20

Expression of the biologically active beta-subunit of mouse nerve growth factor (beta-NGF) was conferred onto cultured AtT-20 mouse pituitary cells via a replication-defective retroviral vector. The retroviral LTR promoter was used for expression of a cDNA for beta-NGF corresponding to the shorter mRNA species produced by most tissues that receive sympathetic innervation. The vector included the TU5 gene conferring resistance to the neomycin analogue G418 under the control of an SV40 early promoter. AtT-20 cells, which produce essentially no endogenous beta-NGF, were infected and then cloned under G418 selection. Clones were evaluated for release into the medium of biologically active beta-NGF using a bioassay for neurite extension from PC-12 cells. The biological activity was equivalent to 1 to 10 ng of beta-NGF per mg cell protein over 24 hours. Immune precipitation and SDS/polyacrylamide gel electrophoresis of labelled proteins in the medium showed that the major form of immunoreactive beta-NGF secreted from cells comigrated with authentic mature beta-NGF, apparent Mr 13,000. Release of this beta-NGF from cells was stimulated by addition of 1 mM-8-bromocyclic AMP or 10 nM-corticotropin releasing factor, suggesting that at least some of the processed factor is stored in secretory vesicles. These studies, together with those on other cultured cells, which produce beta-NGF and lack secretory granules, e.g. L cells, suggest that the beta-NGF precursor synthesized from the shorter mRNA species can be processed and secreted through either the regulated or constitutive route. This retroviral vector provides a potential means of conferring beta-NGF expression onto a number of different cell types in culture and in vivo.