Identification of a murine methyl cytosine phosphate guanine binding domain-containing factor 3 (MBD3) promoter segment sufficient for driving reporter gene expression in neurons in vitro and in vivo.
In this study, we characterize the functional properties of a segment of the murine methyl cytosine phosphate guanine binding domain-containing factor 3 (MBD3) promoter region. Transient transfection of a chimera consisting of a 1072 base pair region extending upstream from the MBD3 initiation codon fused to a luciferase complementary DNA (cDNA) confirmed the presence of a functional promoter unit. Primer extension analysis failed to identify a single predominant transcription initiation site, but rather detected multiple transcription initiation sites in both brain tissue and cultured neuroblastomaxglioma cell line (NG108-15) and rat pheochromocytoma cell line (PC12) cells. Reporter gene assays revealed that this 1072 base pair fragment efficiently drives expression in transfected NG108-15 cells, PC12 cells, cultured primary neurons, and in neurons of a transgenic mouse brain. Deletion analysis mapped the critical region for promoter activity to a segment of approximately 518 base pairs, located from positions -585 to -68 relative to the translational start codon. Taken together, these data indicate that a 1072 base pair fragment of the MBD3 promoter is sufficient to drive expression in cell lines and primary cultured neurons, and is able to direct transgene expression in the mouse brain in a pattern with spatial similarity to that of native MBD3.
Jung
B P
BP
Division of Cellular and Molecular Biology, Toronto Western Research Institute, Mac 11-412, 399 Bathurst Street, Toronto, Ontario, Canada.
Purushotham
D
D
Eubanks
J H
JH
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003360:Cosmids; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016678:Genome; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D004268:DNA-Binding Proteins; C492013:Mbd3 protein, mouse; D014157:Transcription Factors; D008156:Luciferases