Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector.
AUTOR(ES)
Asselbergs, F A
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=147460Documentos Relacionados
- Locus Control Region of the Human CD2 Gene in a Lentivirus Vector Confers Position-Independent Transgene Expression
- Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression.
- Position-independent germline transformation in Drosophila using a cuticle pigmentation gene as a selectable marker.
- Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain.
- Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.