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 growth 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 one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.
Asselbergs
F A
FA
Pharma Research Department, Novartis Pharma Inc., CH-4002 Basel, Switzerland. fred_am.asselbergs@pharma.novartis.com
Grossenbacher
R
R
Ortmann
R
R
Hengerer
B
B
McMaster
G K
GK
Sutter
E
E
Widmer
R
R
Buxton
F
F
D000818:Animals; D001483:Base Sequence; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D007739:L Cells; D008156:Luciferases / Q000096:biosynthesis; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection / Q000379:methods
D017931:DNA Primers; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D003907:Dexamethasone; D008156:Luciferases