POSITION-INDEPENDENT EXPRESSION OF A HUMAN NERVE GROWTH FACTOR-LUCIFERASE REPORTER GENE CLONED ON A YEAST ARTIFICIAL CHROMOSOME VECTOR

Two yeast artificial chromosomes containing the entire human nerve gro wth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to on e of the YAC telomeres, Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulatio n of endogenous mouse NGF protein in mouse L929 fibroblasts, In contra st to the plasmid-based reporter gene, expression and regulation of th e YAC-based reporter gene was independent of the site of integration o f the transgene, Basic fibroblast growth factor and okadaic acid stimu lated expression of the YAC transgene, whereas transforming growth fac tor-p and dexamethasone inhibited it, Although cyclic AMP strongly sti mulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endog enous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, b ut downregulation of the expression of NGF reporter genes occurred onl y at EC50 of 10 nM. This higher concentration was also required for up regulation of luciferase genes driven by the dexamethasone-inducible p romoter of the mouse mammary tumor virus in L929 fibroblasts.